The relative significance of acetate and glucose as precursors for lipid synthesis in liver and adipose tissue from ruminants

1. The incorporation of labelled glucose into lipid by liver slices from sheep and cows is considerably less than that by liver slices from the rat, although oxidation to carbon dioxide occurs to a similar extent. ATP citrate lyase and NADP malate dehydrogenase are inactive in both sheep and cow liver but active in rat liver. The absence of the citrate-cleavage pathway of lipogenesis in ruminant liver has been confirmed by the negligible amounts of C-3 of aspartate incorporated into fatty acids. 2. Considerable amounts of [(14)C]acetate are incorporated into fatty acids and non-saponifiable lipid in rat and ruminant liver. Acetyl-CoA synthetase, the initial enzyme in the metabolism of acetate, has a high activity in liver from rat and ruminants. 3. In adipose tissue from ruminants more acetate than glucose is converted into lipids, whereas the converse is true in rat adipose tissue. The greater incorporation of [(14)C]acetate into fatty acids in adipose tissue from the ruminant as compared with the non-ruminant may be caused, in part, by the higher activity of acetyl-CoA synthetase activity in the ruminant. 4. The results suggest that, in both liver and adipose tissue from ruminants, acetate is a more important source of lipid than glucose. 5. Two enzymes of the hexose monophosphate shunt, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, are active in both tissues and from the three species.     

Studies of the in vivo metabolism of mevalonic acid in the neonatal chick

After 4 hr of the intraperitoneal injection of different doses of (R)-[5-14C]mevalonic acid (MVA), its incorporation into nonsaponifiable and saponifiable lipids was maximal in neonatal chick kidneys and liver, and minimal in brain, spinal cord and skin. Using 14CO2 production from [5-14C]MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that about 11% of MVA was in vivo metabolized by this pathway in nonmammalian species. Kidneys presented the maximal ability to incorporate MVA into nonsaponifiable and saponifiable lipids at any time considered (15-750 min). The percentage of radioactivity recovered as saponifiable lipids in liver and kidney decreased after 12 hr the injection of MVA. Although the absolute amounts of 14C incorporated in both derivatives were much less in brain, spinal cord and skin than in liver and kidneys, the relative percentages found in the saponifiable fraction were clearly higher in the former tissues, especially in the spinal cord.     

Effect of α-p-chlorophenoxyisobutyrate on the metabolism of isoprenoid compounds in the rat

1. Feeding of alpha-p-chlorophenoxyisobutyrate (CPIB) to rats increased ubiquinone concentration in the liver but not in other tissues. The increase was progressive with the time of feeding and related to the concentration of CPIB in the diet. 2. Incorporation of [1-(14)C]acetate, but not of [2-(14)C]mevalonate, into sterols in the liver in vivo or by liver slices in vitro was decreased on feeding the rats with CPIB. However, incorporation of mevalonate into ubiquinone increased. 3. CPIB, when added in low concentrations to liver slices, had no effect on isoprene synthesis from acetate; higher concentrations, however, were inhibitory. 4. No activation of ubiquinone synthesis from mevalonate was observed when CPIB was added to the liver slices synthesizing ubiquinone. 5. The increase in ubiquinone in CPIB-fed animals appears to be due to increased synthesis in the initial stages and to decreased catabolism in the later stages. 6. An inverse relationship was found between the concentration of ubiquinone in the liver and the serum sterol concentration in CPIB-fed rats.     

Contribution of omega-oxidation to fatty acid oxidation by liver of rat and monkey.

Contributions of omega-oxidation to overall fatty acid oxidation in slices from livers of ketotic alloxan diabetic rats and of fasted monkeys are estimated. Estimates are made from a comparison of the distribution of 14C in glucose formed by the slices from omega-14C-labeled compared to 2-14C-labeled fatty acids of even numbers of carbon atoms and from [1-14C]acetate compared to [2-14C]acetate. These estimates are based on the fact that 1) the dicarboxylic acid formed via omega-oxidation of a omega-14C-labeled fatty acid will yield [1-14C]acetate and [1-14C]succinate on subsequent beta-oxidation, if beta-oxidation is assumed to proceed to completion; 2) only [2-14C]acetate will be formed if the fatty acid is metabolized solely via beta-oxidation; and 3) 14C from [1-14C]acetate and [1-14C]succinate is incorporated into carbons 3 and 4 of glucose and 14C from [2-14C]acetate is incorporated into all six carbons of glucose. From the distributions found, the contribution of omega-oxidation to the initial oxidation of palmitate by liver slices is estimated to between 8% and 11%, and the oxidation of laurate between 17% and 21%. Distributions of 14C in glucose formed from 14C-labeled palmitate infused into fasted and diabetic rats do not permit quantitative estimation of the contribution of omega-oxidation to fatty acid oxidation in vivo. However, the distributions found also indicate that, of the fatty acid metabolized by the whole animal in the environment of glucose formation, at most, only a minor portion is initially oxidized via omega-oxidation. As such, omega-oxidation cannot contribute more than a small extent to the formation of glucose.     

Localization of sites of lipid biosynthesis in mammalian epidermis

The end-product of epidermal differentiation is a stratified layer of corneocytes whose extracellular lipid bilayers provide a permeability barrier. It is generally accepted that the epidermis synthesizes most if not all of the lipids found in this tissue and that extra-epidermal tissues contribute very little to this lipid content. Moreover, the individual epidermal strata in which epidermal lipid biosynthesis occurs are not known. To address this question, we examined [3H]H2O incorporation into nonsaponifiable and saponifiable lipids in individual epidermal cell layers 3 hr after intraperitoneal injection into neonatal mice, and compared this to protein and DNA synthesis using intraperitoneal [3H]leucine and [3H]thymidine incorporation, respectively. Lipid biosynthesis was also assessed by [14C]acetate incorporation into lipid fractions in organ cultured skin and in epidermal subpopulations. The in vivo studies demonstrated that the biosynthetic activity of both saponifiable and nonsaponifiable lipids was comparable to, if not greater, in the stratum granulosum (SG) than in basal/spinous (SB + SS) layer, despite significantly lower levels of both protein and DNA synthesis in the SG. On a mass basis, the SG accounts for about four times the biosynthetic activity of the combined SB + SS layers. The lipid biosynthetic activity in vitro also was two- to fivefold higher in the SG, regardless of whether the epidermis was separated into individual cell layers before or after incubations with radiolabel. Moreover, this difference could not be ascribed to increased acetate pools or to elevated metabolism in the SG versus the SB + SS since the rates of CO2 production were much lower in the SG fraction. The increase in lipid biosynthesis in SG over SB + SS was greatest for phospholipids, followed by glycosphingolipids, and free sterols but was observed in almost all lipid classes. These studies show not only that mammalian epidermis is an active site of de novo lipid biosynthesis, but also that this activity remains high in the stratum granulosum, while other forms of metabolic activity are diminishing. These observations are consistent with the knowledge that lipids extruded from the stratum granulosum layer provide the hydrophobic permeability barrier, and further suggest that elevated synthetic activity in the stratum granulosum would allow rapid replenishment in the event that the barrier is damaged.     

Acetate assimilation by the fission yeast, Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose. The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins. Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate. However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe. [1-14C]Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids. Assimilation of [1-14C]acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.     

The role of circulating mevalonate in nephrotic hypercholesterolemia in the rat

The cause of the hypercholesterolemia that characterizes the nephrotic syndrome has never been adequately explained. The present study examines the possibility that enhanced availability of the cholesterol precursor, mevalonic acid, to the liver in the nephrotic state may result in increased hepatic cholesterogenesis. In normal animals, the kidneys are known to be the major site of the metabolism of circulating mevalonate to both cholesterol and CO2. Previous studies, using perfusion of isolated, intact kidneys, have shown that the excretion and metabolism of mevalonate are both impaired in nephrosis. The present investigation has demonstrated in vivo that puromycin aminonucleoside nephrosis results in a 25% reduction in the oxidation of mevalonate to CO2. In the same nephrotic animals, cholesterogenesis from circulating mevalonate was significantly increased in both liver and carcass. In addition, liver slices from nephrotic animals incorporated increased amounts of [5-14C]mevalonate into cholesterol when calculated per whole liver, but not per gram of liver. Oxidation of mevalonic acid by kidney slices was significantly reduced, whether expressed as per gram of tissue or per whole organ. HMG-CoA (3-hydroxy-3-methylglutaryl) reductase activity in liver of nephrotic animals was significantly increased. We conclude that, in the nephrotic state, impaired mevalonate metabolism by the kidney may contribute to enhanced cholesterogenesis by increasing delivery of mevalonate to liver and carcass; in addition, nephrosis appears to provide an undefined stimulus for HMG-CoA reductase activity in the liver, thereby providing an additional enhancement of hepatic cholesterogenesis.     

In vitro metabolism of rumenic acid in bovine liver slices

Ruminant products are the major source of CLA for humans. However, during periods of fat mobilization, the liver might play an important role in CLA metabolism which would limit the availability of the latter for muscles and milk. In this context, rumenic acid (cis-9, trans-11 CLA) metabolism in the bovine liver (n = 5) was compared to that of oleic acid (n = 3) by using the in vitro liver slice method. Liver slices were incubated for 17 h in a medium containing 0.75 mM of FA mixture and 55 microM of either [1-(14)C] rumenic acid or [1-(14)C] oleic acid at 37 degrees C under an atmosphere of 95% O(2)-5% CO(2). Rumenic acid uptake by liver slices was twice (P = 0.009) that of oleic acid. Hepatic oxidation of both FA (> 50% of incorporated FA) led essentially to the production of acid-soluble products and to a lower extent to CO(2) production. Rumenic acid was partly converted (> 12% of incorporated rumenic acid) into conjugated C18:3. CLA and its conjugated derivatives were mainly esterified into polar lipids (71.7%), whereas oleic acid was preferentially esterified into neutral lipids (59.8%). Rumenic acid secretion as part of VLDL particles was very low and was one-fourth lower than that of oleic acid. In conclusion, rumenic acid was highly metabolized by bovine hepatocytes, especially by the oxidation pathway and by its conversion into conjugated C18:3 for which the biological properties need to be elucidated.     

Metabolism of intracerebrally injected mevalonate in brain of suckling and young adult rats

We have investigated the in vivo metabolism via sterol and nonsterol pathways of intracerebrally injected mevalonate (MVA) in brains from suckling (10-day-old) and young adult (60-day-old) rats. Results of our study indicated that increasing the amounts of MVA injected increased MVA incorporation into all the lipid fractions examined. The incorporation of MVA into nonsaponiable lipids (NSF) and digitonin precipitable sterols (DPS) was similar in brains from adult and suckling rats. In brain tissue from both suckling and young adult rats the synthesis of dolichol from MVA varied with the amounts of MVA injected. Significant amounts of MVA were recovered in phosphorylated and free polyprenols (farnesol and geraniol) in brain tissue from rats of both ages. Also in both groups of animals, the amounts of MVA incorporated in phosphorylated and free farnesol were higher than the amounts recovered in either, phosphorylated or free geraniol. The amounts of MVA incorporated into the prenoic/fatty acid fraction by brain tissue from both suckling and young adult rats were less than 1% of the total MVA incorporated (nonsaponifiable and saponifiable lipids). Incorporation of MVA into the prenoic/fatty acid fraction by brain tissue was higher in suckling than in young adult rats. These data indicate that the brain tissue from suckling and young adult rats do not differ in their capacity to metabolize MVA into squalene and sterols and that in brain, metabolism of MVA by a shunt pathway is minimal. This suggests that in vivo regulation of cholesterol synthesis during brain development must occur at a step(s) in the sterol synthetic pathway prior to mevalonate, and that metabolism of mevalonate by shunt pathway did not play a role in the developmental regulation of brain sterol synthesis. The data also suggest that in both groups of animals the synthesis of squalene by synthetase may in part control brain sterol synthesis and the synthesis of dolichol is regulated by MVA concentration in the tissue.     

The isolation of 2,3-oxidosqualene from the liver of rats treated with 1-dodecylimidazole, a novel hypocholesterolaemic agent

1. Non-saponifiable lipid from the livers of rats treated with 1-dodecylimidazole contained an unidentified compound that was not present in the livers from untreated animals. 2. Treated rats had lower serum cholesterol concentrations than control rats. 3. 1-Dodecylimidazole, when added to rat liver slices, inhibited the incorporation of [1-(14)C]acetate and [2-(14)C]mevalonate into digitonin-precipitable sterols and resulted in the accumulation of a labelled compound, which was chromatographically identical with the unknown compound described in 1 above. 4. Rats treated with 1-dodecylimidazole incorporated less [(14)C]mevalonate into liver digitonin-precipitable sterols than untreated animals and accumulated the unknown compound as a labelled intermediate. 5. The unknown intermediate had the same chromatographic properties, n.m.r. and mass spectra as authentic 2,3-oxidosqualene. 6. The identity of the intermediate as 2,3-oxidosqualene was further established by showing that it was incorporated into sterols by rat liver homogenates under anaerobic conditions. In addition, incubation of [(14)C]squalene with rat liver homogenates resulted in trapping of the radioactivity by the added intermediate. 7. It is suggested that the hypocholesterolaemic activity of 1-dodecylimidazole results in part from the inhibition of cholesterol biosynthesis at the level of 2,3-oxidosqualene sterol cyclase.     

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.     

Effect of formate on 14C incorporation from various precursors into rat liver proteins and lipids

Experiments on the rat liver homogenates and slices show that formate stimulates carbon incorporation from [1-14C] lysine and [2-14C] acetate into proteins and from [2-14C] acetate into lipids. The stimulating effect depends on both the formate concentration and nature of the labelled precursor. The in vitro experiments demonstrate the highest stimulating effect on the metabolism of both proteins and lipids under administration of 2 microM formate per 100 g of animal mass. Determination of the label incorporation rate at different time after formate administration showed that the latter evokes an intensified synthesis of protein with rate of its decay remaining the same.     

Rates of cholesterol, ubiquinone, dolichol and dolichyl-P biosynthesis in rat brain slices

Slices from the brain and liver of rats were prepared and upon incubation exhibited a continuous and high capacity for incorporation of radioactive precursors into proteins and lipids. Using [3H]mevalonate as precursor, the rates of biosynthesis of cholesterol, ubiquinone, dolichol and dolichyl-P in brain slices were determined and found to be 5.5, 0.25, 0.0093 and 0.0091 nmol/h/g, respectively. Dolichol and dolichyl-P accumulate to a limited extent, but almost all of these lipids in the brain originate from de novo synthesis. The calculated half-lives for cholesterol, ubiquinone, dolichol and dolichyl-P were 4076, 90, 1006 and 171 h, respectively. The results indicate that lipids formed via the mevalonate pathway in the brain have an active and independently regulated biosynthesis.     

Lipogenesis from ketone bodies in rat brain. Evidence for conversion of acetoacetate into acetyl-coenzyme A in the cytosol.

The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.     

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.     

Lack of adaptation in lipogenesis by hepatoma 9121

The "minimal deviation" hepatoma 9121, implanted in rats, was shown to biosynthesize fatty acids from acetate-1-(14)C at the same rate as normal rat liver but faster than host liver. Feeding the host animals a fat-deficient diet caused fatty acid biosynthesis to be increased 3- to 13-fold in liver, but the dietary regimen did not influence fatty acid biosynthesis in the tumor tissue. Oxygen consumption and the oxidation of acetate and mevalonate to CO(2) were all affected by the dietary manipulation in liver but not in hepatoma. The fat-deficient diet decreased incorporation of acetate and mevalonate into cholesterol by the liver of control animals, increased it in the liver of host animals, and had no effect on this process in hepatoma. Thus, the transplantable tumor has lost the adaptive power of its parent tissue to respond to the dietary stimulus. The changes in fatty acid composition in total lipids in response to the fasting and refeeding were also markedly different in hepatoma from those in liver of the host animals. These results support the concept that this tumor is characterized by a loss of some metabolic controls.     

Fatty acid oxidation and esterification in isolated rat hepatocytes: regulation by dibutyryl adenosine 3',5'-cyclic monophosphate

Isolated rat hepatocytes rapidly utilized [(14)C]palmitate and, in particular, synthesized large amounts of neutral lipids from palmitate. Incorporation into cellular lipids occurred at a linear rate proportional to the medium concentration of fatty acids. Oxidation of [(14)C]palmitate to CO(2) increased with time and was much slower than palmitate esterification. Since [(14)C]acetate and [(14)C]glucose were oxidized to CO(2) at a linear rate, the lag in fatty acid oxidation to CO(2) did not involve enzymatic steps subsequent to acetate formation. The relative contribution of palmitate to esterification and to CO(2) formation depended upon the molar ratio of palmitate to albumin (v) and the length of incubation. Dibutyryl cyclic AMP (1 mM) reduced the oxidation of palmitate and acetate to CO(2) by about 50 and 90%, respectively, but did not alter palmitate esterification. However, equivalent concentrations of sodium butyrate produced similar decreases in CO(2) formation. Dibutyryl cyclic AMP (1 mM) also stimulated palmitate oxidation to water-soluble products, principally ketone bodies, by 50-100%. Sodium butyrate exerted no effect, while monobutyryl cyclic AMP and cyclic AMP both stimulated this pathway significantly. These results indicate that both v and dibutyryl cyclic AMP regulate the metabolism of fatty acids by isolated hepatocytes and suggest that hormonal stimulation of adenyl cyclase controls hepatic lipid metabolism.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

The in vitro metabolism of mevalonate by sterol and non-sterol pathways.

The metabolism of mevalonic acid by both sterol and non-sterol pathways has been evaluated in nine tissues of the rat. An in vitro estimation of the non-sterol, or "shunt", pathway of mevalonate metabolism was made possible by determining the conversion of [2-14C]mevalonate or [5-14C]mevalonate to 14CO2 in tissue slices. In confirmation of our previous results, the kidney was found to play a major role in the metabolism of mevalonate to sterols and sterol precursors. The shunt pathway accounted for a significant percentage of the mevalonate metabolized in kidney, ileum, spleen, lung and testes, but was of minor importance or undetectable in liver, brain, skin, and adipose tissue. Kidney, however, proved to be by far the most active tissue site of mevalonate metabolism by the shunt mechanism in that, on an average, renal tissue metabolized (R)-[14C]mevalonate over the non-sterol pathway at a rate that was 21 times that of any other tissue examined. These results indicate that the kidneys are of major importance in the metabolism of mevalonate by each of the known pathways of metabolism of this sterol precursor.     

Alterations in cholesterol and fatty acid biosynthesis in rat liver homogenates by aryloxy acids (Short Communication)

2,4-Dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid inhibited the incorporation of [2-(14)C]mevalonate into cholesterol and non-saponifiable lipids. Both compounds inhibited the conversion of [1-(14)C]isopentenyl pyrophosphate into cholesterol and the synthesis of cholesterol and fatty acids from [2-(14)C]acetate. There was no inhibition of the conversion of [1-(14)C]mevalonate into CO(2). At low concentrations (0.5mm) of the compounds there was a stimulation of acetate incorporation into fatty acids.