Comparative effects of (−)-hydroxycitrate and (+)-allo-hydroxycitrate on acetyl CoA carboxylase and fatty acid and cholesterol synthesis in vivo

(−)-Hydroxycitrate and (+)-allo-hydroxycitrate were investigated for their effects on lipid synthesis in vivo under conditions of either high carbohydrate feeding or 24 hr fasting. Changes in rates of lipid synthesis resulting from the oral administration of these compounds were monitored with the use of radiolabeled H₂O, alanine, and acetate. In the fed rat, (−)-hydroxycitrate significantly reduced the incorporation of H₂O and alanine into fatty acids and cholesterol. An increased incorporation of labeled H₂O into fatty acids but no change in cholesterol synthesis in the fasted rat suggested that (−)-hydroxycitrate may be an activator of acetyl CoA carboxylase. With (−)-hydroxycitrate administration, acetate incorporation into fatty acids and cholesterol was subject to pool dilution effects under fed or fasted states. (+)-allo-Hydroxycitrate was ineffective in modulating the rates of fatty acid synthesis under either nutritional condition. Both (−)-hydroxycitrate and (+)-allo-hydroxycitrate were shown to be in vitro activators of acetyl CoA carboxylase, the former being a much stronger activator than the latter. Thus, stereospecificity of the hydroxycitrate isomers was demonstrated in both the inhibition of lipid synthesis (previously shown to occur at adenosine triphosphate citrate lyase) and the stimulation of fatty acid synthesis (possibly occurring at acetyl CoA carboxylase).     

Ketone bodies,glucose and glutamine as lipogenic precursors in human diploid fibroblasts

Incorporation of [¹⁴C] from acetoacetate, D(-)- and L(+)-3-hydroxybutyrate, glucose, glutamine, acetate and palmitate in cellular lipids were studied in cultures in human diploid fibroblasts (HDF). The results showed that acetoacetate was 2–10 times more effective as a lipogenic precursor than was either D- or L-3-hydroxybutyrate. Its extent of incorporation into lipids was 2- to 8-fold more than the other precursors examined under conditions when the overall rates of nonsaponifiable and saponi-fiable lipogenesis as measured by³H₂O incorporation were essentially unchanged. Acetoacetate supported both saponifiable and nonsaponifiable lipid syntheses with half-saturation values (K_m app.) of 185 μM and 30 μM, respectively. Glucose stimulated acetoacetate incorporation into lipids whereas, conversely, acetoacetate inhibited [¹⁴C] glucose incorporation into lipids. The presence of low density lipoproteins (LDL) cholesterol (@40 μg cholesterol/mL) inhibited the incorporation of [¹⁴C] from acetoacetate 56% into nonsaponifiable lipids; the inhibition was consistently higher (75%) when [¹⁴C] glucose or glutamine were the precrusors. The loss of 3-hydroxy-3-methyl-glutaryl CoA (HMG CoA) reductase activity upon addition of LDL-cholesterol was greater than the suppression of [¹⁴C] incorporation from acetoacetate or glucose into nonsaponifiable lipids. In the presence of glucose, [¹⁴C] acetoacetate was incorporated into 3-βOH sterols (digitonin precipitable). 7.7±1.1 times more effectively than was [¹⁴C] glucose. The results suggest that HDF would be a suitable model to investigate the effects of various precrusors of HMG CoA on the rate of cholesterol biosynthesis.     

Partition of ketone bodies into cholesterol and fatty acids in vivo in different brain regions of developing rats

The proportions of labeled ketone bodies and glucose incorporated into cholesterol and fatty acids in different regions of the brain in developing rats were compared. In cerebrums of 15- and 18-day-old rats, the ratios of dpm cholesterol/dpm fatty acids incorporated from [3-¹⁴C] acetoacetate and [3-¹⁴C] β-hydroxybutyrate ranged from 0.4 to 0.7, or 50 to 100% higher than values obtained with [U-¹⁴C] glucose. Much higher ratios were obtained with younger animals: from 1 to 12 days of life, the values ranged from 1.0 to 1.3 with [3-¹⁴C] β-hydroxybutyrate as substrate, and, from 1 to 5 days, with [3-¹⁴C] acetoacetate, they were 1.0 or greater. During the first 12 days of life, the ratios resulting from administration of [U-¹⁴C] glucose were 0.4–0.7. Clearly, a greater proportion of acetoacetate and β-hydroxybutyrate was incorporated into cholesterol during the first week of life than the remaining suckling period. Like cerebrum, other brain regions (i.e., cerebellum, midbrain, brain stem and thalamus) yielded higher ratios of dpm cholesterol/dpm fatty acids from [3-¹⁴C] β-hydroxybutyrate during the first 12 days of life than on day 17. Brain stem was the most active region for lipid synthesis, and had the highest dpm cholesterol/dpm fatty acid ratio. Since active synthesis of cholesterol from ketone bodies during the early postnatal period coincides with a period of rapid brain growth, the results indicate that ketone bodies are more important early in the suckling period as sources of cholesterol for brain growth.     

Pulmonary surfactant lipid synthesis from ketone bodies,lactate and glucose in newborn rats

The contribution of acetoacetate (AcAc), β-hydroxybutyrate (βOHB), lactate and glucose to pulmonary surfactant lipid synthesis in three-to five-day-old rats was measured. Minced lung tissue was incubated with³H₂O and [3-¹⁴C]AcAc, [3-¹⁴C]βOHB, [U-¹⁴C]lactate or [U-¹⁴C]glucose, and the radioactivity incorporated into surfactant lipids was measured. When expressed as nmol of substrate incorporated/g lung tissue per four hr, lactate was incorporated more rapidly than other substrates into total surfactant lipids and phosphatidylcholine (PC). There was no difference in the rates of incorporation of lactate, AcAc or glucose into disaturated PC (DSPC). Substrates other than glucose were incorporated almost exclusively into fatty acids, whereas 60–80% of glucose incorporated into surfactant phospholipids was found in fatty acids, with the remaining in glyceride-glycerol. When expressed as nmol acetyl units incorporated/g lung tissue per four hr, the rates of AcAc, lactate and glucose incorporation into total surfactant fatty acids were comparable. Glucose incorporation into DSPC and PC was greater than that of AcAc and lactate. When glucose was the only exogenous substrate added to the incubation medium, it contributed 37% of total surfactant fatty acids synthesized de novo. In the presence of other substrates, the contribution of glucose to de novo fatty acid synthesis dropped to 14–20%. In the presence of unlabeled glucose,¹⁴C-labeled AcAc, lactate and βOHB contributed 52%, 40% and 19%, respectively, of the total fatty acids synthesized de novo. The rate of βOHB incorporation into surfactant lipids was only about 50% that of other substrates and was accompanied by low activity of β-hydroxybutyrate dehydrogenase measured for newborn lung. These results demonstrate that AcAc and lactate are important precursors for surfactant lipids in neonatal rat lung.     

Lipoprotein lipid and protein synthesis in experimental nephrosis and plasmapheresis. I: Studies in rat in vivo

The incorporation of L-4,5-[³H]leucine into the ultracentrifugally separated apolipoproteins of very low, low, and high density lipoproteins (VLDL, LDL, HDL) and into serum albumin was found three-to four-fold higher in nephrotic than in normal rats one hour after intravenous injection. Incorporation of leucine into the circulating lipids was negligible. Increases of similar magnitude were obtained in the incorporation of simultaneously injected 1,5[¹⁴C] citrate into the lipids of VLDL, LDL, and HDL of nephrotic rats. Of the citrate carbons incorporated into serum and liver lipids, the proportion in cholesterol was higher in nephrotic rats when compared to normal rats. The incorporation of both precursors into total proteins and lipids of the liver vs. the incorporation into the lipoproteins was relatively lower in nephrotic than in control rats, indicating a preferential channeling into secretable products. The occurrence of enhanced new lipid synthesis in nephrosis was corroborated by the finding of markedly enhanced synthesis of lipoprotein-borne fatty acids and cholesterol from³H₂O. These results point out that while leucine is not an efficient in vivo precursor of lipoprotein lipids in nephrosis, de novo lipogenesis proceeds from other precursors. Similar trend of changes, though of smaller magnitude, was elicited in rats after double plasmapheresis, 18 hr apart, when measured 3 hr after the second plasma withdrawal. This indicates that the loss of circulating proteins either by direct removal or through kidney lesion stimulates the compensatory hepatic response involving excessive lipoprotein synthesis. Time-course studies showed that peak incorporation of leucine and citrate into the protein and lipid components of lipoproteins, respectively, as well as into serum albumin, occurred coincidentally 3 hr after the second plasmapheresis, suggesting an interdependence of the enhanced protein and lipid synthesis.     

Stearic acid unlike shorter-chain saturated fatty acids is poorly utilized for triacylglycerol synthesis and β-oxidation in cultured rat hepatocytes

Utilization of stearate as compared to various saturated fatty acids for cholesterol and lipid synthesis and β-oxidation was determined in primary culture of rat hepatocytes. At 0.5 mmol/L in the medium, stearate (18:0) adequately solubilized by albumin was less inhibitory to cholesterol synthesis from [2-¹⁴C] acetate than myristate (14:0) and palmitate (16:0) (68% vs. 91 and 88% inhibition, respectively). The rate of incorporation into cholesterol from [1-¹⁴C] stearate (3.0±0.6 nmol/mg protein/4 h) was 37-, 1.8-, and 7.8-fold of that from myristate, palmitate, and oleate, respectively. Conversely, the rate of [1-¹⁴C] stearate incorporation into total glycerolipids was 88–90% lower than that of labeled palmitate, myristate, and oleate. The rate of [1-¹⁴C] stearate incorporation into triacylglycerol (3.6±0.4 nmol/mg protein/4 h) was 6–8% of that from myristate, palmitate, oleate, and linoleate. The rate of stearate incorporation into phospholipids was the lowest among tested fatty acids, whereas the rate of mono- and diacylglycerol synthesis was the highest with stearate treatment. The rate of β-oxidation as measured by CO₂ and acid soluble metabolite production was also the lowest with [1-¹⁴C] stearate treatment at 22.7 nmol/mg protein/4 h, which was 35–40% of those from other [1-¹⁴C] labeled fatty acids. A greater proportion of stearate than other fatty acids taken up by the hepatocytes remained free and was not metabolized. Clearly, stearate as compared to shorter-chain saturated fatty acids was less efficiently oxidized and esterified to triacylglycerol in cultured rat hepatocytes.     

Relative utilization of fatty acids for synthesis of ketone bodies and complex lipids in the liver of developing rats

The regulation of hepatic ketogenesis, as related to the metabolism of fatty acids through oxidative and synthetic pathways, was studied in developing rats. [1-¹⁴C] palmitate was used as a substrate to determine the proportions of free fatty acids utilized for the production of ketone bodies, CO₂ and complex lipids. Similar developmental patterns of hepatic ketogenesis were obtained by measuring the production of either [¹⁴C]acetoacetate from exogenous [1-¹⁴C] palmitate or the sum of unlabeled acetoacetate and β-hydroxybutyrate from endogenous fatty acids. The production of total ketone bodies was low during the late fetal stage and at birth, but increased rapidly to a maximum value within 24 hr after birth. The maximal ketogenic capacity appeared to be maintained for the first 10 days of life.¹⁴CO₂ production from [1-¹⁴C] palmitate increased by two- to fourfold during the suckling period, from its initial low rate seen at birth. The capacity for synthesis of total complex lipids was low at birth and had increased by day 3 to a maximal value, which was comparable to that of adult fed rats. The high lipogenic capacity lasted throughout the remaining suckling period. When ketogenesis was inhibited by 4-pentenoic acid, the rate of synthesis of complex lipids did not increase despite an increase in unutilized fatty acids. During the mid-suckling period, approximately equal amounts of [1-¹⁴C] palmitate were utilized for the synthesis of ketone plus CO₂ and for complex lipid synthesis. By contrast, in adult fed rats, the incorporation of fatty acids into complex lipids was four times higher than that of ketone plus CO₂. These observations suggest that stimulated hepatic ketogenesis in suckling rats results from the rapid oxidation of fatty acids and consequent increased production of acetyl CoA, but not from impaired capacity for synthsis of complex lipids.     

Fatty acid and sterol synthesis by rat small intestine in vitro

Slices of rat jejunum were incubated with [2-¹⁴C]pyruvate, [1-¹⁴C]acetate, or [³H]H₂O to determine lipogenic activity. Under all conditions studied, pyruvate acted as a better precursor than acetate for fatty acid synthesis but not for the synthesis of sterol. Exogenous glucose significantly (P≤0.05) increased the conversion of both pyruvate and acetate to fatty acids. By contrast, fasting resulted in a decrease (p≤0.05) in lipogenic activity. The highest levels of lipogenesis were observed when [³H]H₂O + glucose at a concentration of 20 mM was used. From such experiments, the absolute rate of fatty acid synthesis in the tissue preparation was calculated: 734±54 nmoles acetyl units incorporated into fatty acids/g tissue/hr.     

Garlic reduces plasma lipids by inhibiting hepatic cholesterol and triacylglycerol synthesis

Prompted by the reported hypolipidemic activity of garlic, the present study was undertaken to elucidate the mechanism(s) underlying the cholesterol-lowering effects of garlic. Rat hepatocytes in primary culture were used to determine the short-term effects of garlic preparations on [1-¹⁴C]acetate and [2-³H]glycerol incorporation into cholesterol, fatty acids and glycerol lipids. When compared with the control group, cells treated with a high concentration of garlic extracts [i.e., petroleum ether- (PEF), methanol- (MEF) and water-extractable (WEF) fractions from fresh garlic] showed decreased rates of [1-¹⁴C]acetate incorporation into cholesterol (by 37–64%) and into fatty acids (by 28–64%). Kyolic containingS-allyl cysteine and organosulfur compounds inhibited cholesterogenesis in a concentration dependent manner with a maximum inhibition of 87% at 0.4 mM. At this concentration, Kyolic decreased [1-¹⁴C]acetate incorporation into fatty acids by 67%.S-allyl cysteine at 2.0 and 4.0 mM inhibited cholesterogenesis by 20–25%. PEF, MEF and WEF depressed the rates of [2-³H]glycerol incorporation into triacylglycerol, diacylglycerol and phospholipids in the presence of acetate, but not in the presence of oleate. The results suggest that the hypocholesterolemic effect of garlic stems, in part, from decreased hepatic cholesterogenesis, whereas the triacylglycerol-lowering effect appears to be due to inhibition of fatty acid synthesis. Primary hepatocyte cultures as used in the present study have been proven useful as tools for screening the anticholesterogenic properties of garlic principles.     

Polyenoic acid metabolism in cultured human skin fibroblasts

The incorporation of [1-¹⁴C]linoleic acid, and [1-¹⁴C]linoleic acid into cellular lipids of cultured human skin fibroblasts was studied. Cultured cells took up both labeled fatty acids at nearly the same rate and incorporated them into a variety of lipid classes. At the end of 1 hr incubation with [1-¹⁴C]linoleic acid, radioactivity was found in the triacylglycerol (TG) and choline phosphoglyceride (CPG) pools preferentially. Incorporation into the TG fraction decreased rapidly, while the uptake into CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) fractions increased progressively with longer incubation times. Similar results were obtained with [1-¹⁴C]linoleic acid as precursor. At the end of 24 hr, desaturation and chain elongation of 18∶3 n−3 was more extensive than conversion of 18∶2 n−6 to higher polyenoic acids. During pulse-chase experiments with either fatty acid precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG and CPG fractions, but continued to increase in the SPG and EPG pools. The similar labeling pattern of cellular phospholipids with linoleic or linolenic acids, and data from pulse-chase studies suggest that a direct transfer of fatty acids from CPG to EPG is a likely pathway in fibroblast cultures. Incorporation into the EPG pool during the pulse-chase experiments paralleled extensive desaturation and elongation of linoleic acid into 20∶4 n−6, and 22∶4 n−6; and of linolenic acid into 22∶5 n−3 and 22∶6 n−3.     

Lipogenesis in the developing brain from intracranially administered [1-14C] acetate and [U-14C] glucose

Fifteen-day-old rats divided into two groups were given [1-¹⁴C]acetate or [U-¹⁴C] glucose by intracranial injection and were sacrificed after 1 hr. Analysis of lipids from the two groups showed differences in the incorporation of radioactivity in the polar lipids and cholesterol. Analysis of brain fatty acid showed that whereas radioactivity from acetate was incorporated into saturated, monoand polyunsaturated fatty acids, the radioactivity from [U-¹⁴C] glucose was found only in 16∶0, 18∶0, and 18∶1. No radioactivity was found in polyunsaturated fatty acids even after concentration of this fraction by AgNO₃:SiO₂ thin layer chromatographic method. This difference is discussed in hypothetical terms of nonhomogeneous acetyl CoA pool, formation of acetyl CoA from glucose exclusively inside the mitochondria, and activation of injected acetate to acetyl CoA.     

Incorporation and distribution of saturated and unsaturated fatty acids into nuclear lipids of hepatic cells

Liver nuclear incorporation of stearic (18∶0), linoleic (18∶2n−6), and arachidonic (20∶4n−6) acids was studied by incubation in vitro of the [1-¹⁴C] fatty acids with nuclei, with or without the cytosol fraction at different times. The [1-¹⁴C] fatty acids were incorporated into the nuclei as free fatty acids in the following order: 18∶0>20∶4n−6≫18∶2n−6, and esterified into nuclear lipids by an acyl-CoA pathway. All [1-¹⁴C] fatty acids were esterified mainly to phospholipids and triacylglycerols and in a minor proportion to diacylglycerols. Only [1-¹⁴C] 18∶2n−6-CoA was incorporated into cholesterol esters. The incorporation was not modified by cytosol addition. The incorporation of 20∶4n−6 into nuclear phosphatidylcholine (PC) pools was also studied by incubation of liver nuclei in vitro with [1-¹⁴C]20∶4n−6-CoA, and nuclear labeled PC molecular species were determined. From the 15 PC nuclear molecular species determined, five were labeled with [1-¹⁴C]20∶4n−6-CoA: 18∶0–20∶4, 16∶0–20∶4, 18∶1–20∶4, 18∶2–20∶4, and 20∶4–20∶4. The highest specific radioactivity was found in 20∶4–20∶4 PC, which is a minor species. In conclusion, liver cell nuclei possess the necessary enzymes to incorporate exogenous saturated and unsaturated fatty acids into lipids by an acyl-CoA pathway, showing specificity for each fatty acid. Liver cell nuclei also utilize exogenous 20∶4n−6-CoA to synthesize the major molecular species of PC with 20∶4n−6 at the sn-2 position. However, the most actively synthesized is 20∶4–20∶4 PC, which is a quantitatively minor component. The labeling pattern of 20∶4–20∶4 PC would indicate that this molecular species is synthesized mainly by the de novo pathway.     

Incorporation of radioactive polyunsaturated fatty acids into liver and brain of developing rat

The incorporation of radioactivity from orally administered linoleic acid-1-¹⁴C, linolenic acid-1-¹⁴C, arachidonic acid-³Hg, and docosahexaenoic acid-¹⁴C into the liver and brain lipids of suckling rats was studied. In both tissues, 22 hr after dosing, 2 distinct levels of incorporation were observed: a low uptake (from 18∶2-1-¹⁴C and 18∶3-1-¹⁴C) and a high uptake (from 20∶4-³H₈ and 22∶6-¹⁴C). In adult rats, the incorporation of radioactivity into brain lipids from 18∶2-1-¹⁴C and 20∶4-³H was considerably lower than the incorporation into the brains of the young rats. In the livers of the suckling rats, the activity from the 18 carbon acids was associated mostly with the triglyceride fraction, whereas the activity from the 20∶4-³H₈ and 22∶6-¹⁴C was concentrated in the phospholipid fraction. In the brain lipids, the activity from the different fatty acids was associated predominantly with the phospholipids. In the liver and brain phospholipid fatty acids, some of the activity in the 18∶2-1-¹⁴C and 18∶3-1-¹⁴C experiments was associated with 20 and 22 carbon polyunsaturated fatty acids; however, radioactivity from orally administered 20∶4-³H₈ and 22∶6-¹⁴C was incorporated intact into the tissue phospholipid to a much greater extent compared with the incorporation of radioactivity into 20∶4 and 22∶6 in the experiments where 18∶2-1-¹⁴C and 18∶3-1-¹⁴C, respectively, were administered. Possible reasons for these differences are discussed. Rat milk contains a wide spectrum of polyunsaturated fatty acids, including linoleate, linolenate, arachidonate, and docosahexaenoate. During the suckling period in the rat, there is a rapid deposition of 20∶4 and 22∶6 in the brain. The results of the present experiments suggested that dietary 20∶4 and 22∶6 were important sources of brain 20∶4 and 22∶6 in the developing rat.     

Fatty acid metabolism in marine fish: Low activity of fatty acyl Δ5 desaturation in gilthead sea bream (Sparus aurata) cells

Marine fish have an absolute dietary requirement for C₂₀ and C₂₂ highly unsaturated fatty acids. Previous studies using cultured cell lines indicated that underlying this requirement in marine fish was either a deficiency in fatty acyl Δ5 desaturase or C_18–20 elongase activity. Recent research in turbot cells found low C_18–20 elongase but high Δ5 desaturase activity. In the present study, the fatty acid desaturase/elongase pathway was investigated in a cell line (SAF-1) from another carnivorous marine fish, sea bream. The metabolic conversions of a range of radiolabeled polyunsaturated fatty acids that comprised the direct substrates for Δ6 desaturase ([1-¹⁴C]18∶2n−6 and [1-¹⁴C]18∶3n−3), C_18–20 elongase ([U-¹⁴C]18∶4n−3), Δ5 desaturase ([1-¹⁴C]20∶3n−6 and [1-¹⁴C]20∶5n−3), and C_20–22 elongase ([1-¹⁴C]20∶4n−6 and [1-¹⁴C]20∶5n−3) were utilized. The results showed that fatty acyl Δ6 desaturase in SAF-1 cells was highly active and that C_18–20 elongase and C_20–22 elongase activities were substantial. A deficiency in the desaturation/elongation pathway was clearly identified at the level of the fatty acyl Δ5 desaturase, which was very low, particularly with 20∶4n−3 as substrate. In comparison, the apparent activities of Δ6 desaturase, C_18–20 elongase, and C_20–22 elongase were approximately 94-, 27-, and 16-fold greater than that for Δ5 desaturase toward their respective n−3 polyunsaturated fatty acid substrates. The evidence obtained in the SAF-1 cell line is consistent with the dietary requirement for C₂₀ and C₂₂ highly unsaturated fatty acids in the marine fish the sea bream, being primarily due to a deficiency in fatty acid Δ5 desaturase activity.     

Metabolism of [1-14C]docosahexaenoate (22∶6n−3), [1-14C]eicosapentaenoate (20∶5n−3) and [1-14C]linolenate (18∶3n−3) in brain cells from juvenile turbotScophthalmus maximus

The incorporation of [1-¹⁴C]18∶3n−3, (LNA) and [1-¹⁴C]-22∶6n−3 (DHA), and the metabolismvia the desaturase/elongase pathways of [1-¹⁴C]LNA, and [1-¹⁴C]20∶5n−3 (EPA) were studied in brain cells from newly-weaned (1-month-old) and 4-month-old turbot. The rank order of the extent of net incorporation of both LNA and DHA into glycerophospholipids was total diradyl glycerophosphocholines (CPL)> total diradyl glycerophosphoethanolamines (EPL)> phosphatidylserine (PS) and phosphatidylinositol (PI) and was independent of the polyunsaturated fatty acid added, the age of the fish and the time of incubation. However, the rate of incorporation of LNA into total lipid, CPL, EPL and PS was significantly greater than the rate of incorporation of DHA, and there was a significantly greater amount of DHA incorporated into EPL than LNA. There was no significant difference between the amounts of LNA and DHA incorporated into total lipid, CPL, PS and PI. Therefore, little preferential uptake and incorporation of DHA into brain cells was apparent. In 24-h incubations, on average 1.1% and 8.5% of radioactivity from [1-¹⁴C]LNA and [1-¹⁴C]EPA, respectively, were recovered in the DHA fraction. Therefore, LNA cannot contribute significantly to brain DHA levels in the turbot but EPA can. There were no significant differences between the amounts of radioactivity from either [1-¹⁴C]LNA or [1-¹⁴C]EPA recovered in the individual products/intermediates of the desaturase pathways in brain cells from 30-day-old and 120-day-old turbot.     

Influence of medium-chain triglycerides on lipid metabolism in the chick

The effect of corn oil, coconut oil, and medium-chain triglyceride (MCT, a glyceride mixture consisting almost exclusively of fatty acids of 8 and 10 carbons in length) ingestion on lipid metabolism was studied in chicks. In chicks fed cholesterol-free diets, MCT ingestion elevated plasma total lipids and cholesterol and depressed liver total lipids and cholesterol when compared to chicks receiving the corn oil diet. As a consequence of the opposite effects of MCT ingestion on plasma and liver cholesterol and total lipids, the plasma-liver cholesterol pool was not altered. When cholesterol was included in the diets, dietary MCT depressed liver and plasma total lipids and cholesterol as compared with corn oil, consequently also lowered the plasmaliver cholesterol pool. The in vitro cholesterol and fatty acid synthesis from acetate-1-¹⁴C was higher in liver slices from chicks fed MCT than in those from chicks fed corn oil. The percentage of radioactivity from acetate-1-¹⁴C incorporated into the carboxyl carbon of fatty acids by liver slices was not altered by MCT feeding, indicating that the increased acetate incorporation represented de novo fatty acid synthesis. The conversion of palmitate-1-¹⁴C to C₁₈ acids was increased in liver of chicks fed MCT, implying that fatty acid chain elongating activity was also increased. Studies on the conversion of stearate-2-¹⁴C to mono- and di-unsaturated C₁₈ acids showed that hepatic fatty acid desaturation activity was enhanced by MCT feeding. Data are presented on the plasma and liver fatty acid composition of chicks fed MCT-, corn oil-, or coconut oil-supplemented diets. The principles of laboratory animal care, as promulgated by the National Society for Medical Research, were observed.     

Metabolism of n−3 and n−6 fatty acids in Atlantic salmon liver: Stimulation by essential fatty acid deficiency

Oxidation, esterification, desaturation, and elongation of [1-¹⁴C]18∶2n−6 and [1-¹⁴C]18∶3n−3 were studied using hepatocytes from Atlantic salmon (Salmo salar I.) maintained on diets deficient in n−3 and n−6 polyunsaturated fatty acids (PUFA) or supplemented with n−3 PUFA. For both dietary groups, radioactivity from 18∶3n−3 was incoporated into lipid fractions two to three times faster than from 18∶2n−6, and essential fatty acids (FFA) deficiency doubled the incorporation. Oxidation to CO₂ was very low and was independent of substrate or diet, whereas oxidation to acid-soluble products was stimulated by EFA deficiency. Products from 18∶2n−6 were mainly 18∶3n−6, 20∶3n−6, and 20∶4n−6, with minor amounts of 20∶2n−6 and 22∶5n−6. Products from 18∶3n−3 were mainly 18∶4n−3, 20∶5n−3, and 22∶6n−3, with small amounts of 20∶3n−3. The percentage of 22∶6n−3 in the polar lipid fraction of EFA-deficient hepatocytes was fourfold higher than in n−3 PUFA-supplemented cells. This correlated well with our other results obtained after abdominal injection of [1-¹⁴C]18∶3n−3 and [1-¹⁴C]18∶2n−6. In hepatocytes incubated with [4,5-³H]-22∶6n−3, 20∶5n−3 was the main product. This retrocon-version was increased by EFA deficiency, as was peroxisomal β-oxidation activity. This study shows that 18∶2n−6 and 18∶3n−3 can be elongated and desaturated in Atlantic salmon liver, and that this conversion and the activity of retroconversion of very long chain PUFA is markedly enhanced by FFA deficiency.     

Elongation predominates over desaturation in the metabolism of 18∶3n−3 and 20∶5n−3 in turbot (Scophthalmus maximus) brain astroglial cells in primary culture

The origin of docosahexaenoic acid (DHA, 22∶6n−3) that accumulates in turbot brain during development was investigated by studying the incorporation and metabolismvia the desaturase/elongase pathways of [1-¹⁴C]-labelled polyunsaturated fatty acids (PUFA) in primary cultures of brain astrocytic glial cells. There was little specificity evident in the total incorporation of PUFAs into the turbot astrocytes. However, specificity was apparent in the distribution of the various PUFAs among the individual lipid classes. In particular, there was very specific incorporation of [¹⁴C]arachidonic acid (AA, 20∶4n−6) into phosphatidylinositol balanced by a lower incorporation of this acid into total diradyl glycerophosphocholines. [¹⁴C]-Linolenic acid (LNA, 18∶3n−3) and [¹⁴C]eicosapentaenoic acid (EPA, 20∶5n−3) were metabolizedvia the desaturase/elongase pathways to a significantly greater extent than [¹⁴C]linoleic acid (18∶2n−6) and [¹⁴C]AA. The turbot astrocytes expressed very little Δ5 desaturase activity and only low levels of Δ4 desaturation activity. Although the percentages were small, approximately 4–5 times as much labelled DHA was produced from [¹⁴C]EPA compared with [¹⁴C]LNA. However, it was concluded that very little DHA in the turbot brain could result from the metabolism of LNA and EPA in astrocytic glial cells.     

Biosynthesis of hydrocarbons by algae: Decarboxylation of stearic acid to N-heptadecane inAnacystis nidulans determined by13C- and2H-labeling and13C nuclear magnetic resonance

The distribution of isotopic labels inn-heptadecane enriched from [1,2-¹³C] and [2-¹³C, 2-²H₃) acetates byAnacystis nidulans has been determined by¹³C nuclear magnetic resonance (¹³C NMR). Labeling with [1,2-¹³C] acetate is consistent with assembly from¹³C−¹³C units derived from an acetate “starter” group and 8 malonate units, as in fatty acid biosynthesis, followed by production of a methyl group through bond cleavage of the terminal¹³C−¹³C unit. A comparison of the hydrocarbon with palmitic acid (the only fatty acid produced in sufficient amount for NMR analysis) enriched from [2-¹³C,2-²H₃]acetate by the same culture shows that they have retained the same fraction of²H at corresponding sites, and have therefore undergone identical biosynthetic and hydrogen-deuterium exchange processes, as would be expected ifn-heptadecane originates from de novo-synthesized stearic acid. NRCC No. 18251.     

Labeled oxidation products from [1-14C], [U-14C] and [16-14C]-palmitate in hepatocytes and mitochondria

When [1-¹⁴C], [U-¹⁴C], and [16-¹⁴C]palmitate were oxidized by isolated rat hepatocytes, there was a differential distribution of label as a percent of total oxidized products, such that¹⁴CO₂ from [1-¹⁴C]>[U-¹⁴C]>[16-¹⁴C]-palmitate and acid-soluble radioactivity from [16-¹⁴C]>[U-¹⁴C]>[1-¹⁴C]palmitate. The oxidation of [2,3-¹⁴C]succinate to¹⁴CO₂ by isolated hepatocytes was only 9.1% of that from [1,4-¹⁴C]succinate, demonstrating that the differences in distribution of labeled products are in part due to less¹⁴CO₂ production from label in the even carbon positions entering the citric acid cycle. Apparent total ketone body production from [16-¹⁴C]palmitate was markedly higher than [1-¹⁴C], and [U-¹⁴C]palmitate. In addition, the¹⁴C-acetone:¹⁴CO₂ ratio derived from decarboxylation of labeled acetoacetate from [1-¹⁴C]palmitate was less than 1 and positively correlated to the rate of fatty acid oxidation in hepatocytes. These findings indicate that the known preferential incorporation of the omega-C₂ unit of fatty acids into¹⁴C-ketone bodies also contributed to the differential distribution of labeled products and that this contribution was greatest at the lower rates of fatty acid oxidation. In isolated mitochondria, the distribution of label to¹⁴CO₂ and acid-soluble radioactivity from [1-¹⁴C], [U-¹⁴C] and [16-¹⁴C]palmitate was qualitatively similar to that seen with hepatocytes. The distribution of label from [1-¹⁴C]acetylcarnitine to¹⁴CO₂ and¹⁴C-ketone bodies by mitochondria was identical to that observed from [1-¹⁴C]palmitate, indicating that the higher rates of¹⁴CO₂ production from [1-¹⁴C]palmitate cannot be explained by a preferential oxidation in the citric acid cycle of either extramitochondrial acetyl-CoA (generated in peroxisomes) or the carboxyl terminal of the fatty acid. As shown by others in cell-free systems, we observed that the total oxidation of [16-¹⁴C]palmitate by hepatocytes and mitochondria was significantly less than [1-¹⁴C] and [U-¹⁴C]palmitate, suggesting either incomplete mitochondrial β-oxidation or incomplete degradation of peroxisomal oxidation products. The data indicate that this incomplete oxidation does not, however, contribute to the differential distribution of label to oxidized products.