Studies in lipogenesis in vivo: Fatty acid and cholesterol synthesis during starvation and re-feeding

1. Lipogenesis in vivo has been studied in mice given a 250mg. meal of [U-¹⁴C]glucose (2·5μc) or given an intraperitoneal injection of 25μg. of [U-¹⁴C]glucose (2·0μc). 2. The ability to convert a [U-¹⁴C]glucose meal into fatty acid was not significantly depressed by 6–7hr. of starvation. In contrast, incorporation of ¹⁴C into fatty acid in the liver after the intraperitoneal dose of [¹⁴C]glucose was depressed by 80% and by more than 90% by 1 and 2hr. of starvation respectively. Carcass fatty acid synthesis from the [U-¹⁴C]glucose meal was not depressed by 12hr. of starvation, whereas from the tracer dose of [U-¹⁴C]glucose the depression in incorporation was 80% after 6hr. of starvation. 3. Re-feeding for 3 days, after 3 days' starvation, raised fatty acid synthesis and cholesterol synthesis in the liver fivefold and tenfold respectively above the levels in non-starved control mice. These increases were associated with an increased amount of both fatty acid and cholesterol in the liver. 4. After 18hr. of starvation incorporation of a [U-¹⁴C]glucose meal into carcass and liver glycogen were both increased threefold.     

Studies on lipogenesis in vivo: Fatty acid and cholesterol synthesis in hyperglycaemic-obese mice

1. Lipogenesis has been studied in intact genetically obese mice by measuring the incorporation of a single oral dose of 250mg. of [U-¹⁴C]glucose into fatty acid and cholesterol in the liver and extrahepatic tissues. Studies were also carried out with [U-¹⁴C]glucose added to the diet and fed for 24hr. With either method of isotope administration, the conversion of [U-¹⁴C]glucose into fatty acid was greatly elevated in the livers of the obese mice. In contrast, conversion of the single dose of [¹⁴C]glucose into fatty acid in extrahepatic tissues of obese mice was only half that occurring in the non-obese litter mates. When [¹⁴C]glucose was given in the diet for 24hr. the total accumulation of labelled fatty acid in extrahepatic tissues of obese mice was slightly less than in the non-obese. Uptake of labelled glucose and conversion into fatty acid in adipose tissue of the obese mice decreased with age. 2. Conversion of the single dose of [¹⁴C]glucose into liver cholesterol was comparable in obese and non-obese mice fed on a purified low-fat diet. However, obese mice given this diet for 12 weeks accumulated 1·54% of cholesterol in the liver compared with 0·29% in the non-obese litter mates. This accumulation apparently resulted from a decrease in removal of cholesterol from the liver, rather than an increased synthesis. 3. Conversion of the single dose of [¹⁴C]glucose into extrahepatic fatty acid was decreased by 18hr. starvation proportionally as much in obese as in non-obese mice. The decrease in liver fatty acid synthesis caused by starvation also was considerable in obese mice, although somewhat less marked than in the non-obese. 4. The metabolic derangements in the liver could be more fundamental to the development of the obesity than the changes seen in extrahepatic tissues.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.     

Role of an acidic compartment in synthesis of disaturated phosphatidylcholine by rat granular pneumocytes

A possible role for an acidic subcellular compartment in biosynthesis of lung surfactant phospholipids was evaluated with granular pneumocytes in primary culture. Incubation with chloroquine (100μm) was used to perturb this compartment. With control cells, incorporation of [9,10-³H]palmitic acid into total lipids and into total phosphatidylcholines increased linearly with time up to 4h. Total incorporation into phosphatidylcholine during a 1h incubation was 999+85pmol of [9,10-³H]palmitic acid, 458±18pmol of [1-¹⁴C]oleic acid and 252±15pmol of [U-¹⁴C]glucose per μg of phosphatidylcholine phosphorus. The cellular content of either disaturated phosphatidylcholine or total phosphatidylcholines did not change during a 2h incubation with chloroquine. In the presence of chloroquine, the specific radioactivity of [³H]palmitic acid in disaturated phosphatidylcholine increased by 40%, and that of disaturated-phosphatidylcholine fatty acids from [U-¹⁴C]glucose increased by 125%. Incorporation of [1-¹⁴C]oleic acid into phosphatidylcholine was decreased by chloroquine by 79% and 33% in the presence or absence of palmitic acid respectively. Chloroquine stimulated phospholipase activity in intact cells, and in sonicated cells at pH4.0, but not at pH8.5. The observations indicate that chloroquine stimulates synthesis of disaturated phosphatidylcholine in granular pneumocytes from fatty acids, both exogenous and synthesized de novo, which can be due to stimulation of acidic phospholipase. This stimulation of acidic phospholipase A activity by chloroquine appears to be coupled to the synthesis of disaturated phosphatidylcholine, thereby enhancing remodelling of phosphatidylcholine synthesized de novo. Our findings, therefore, implicate the involvement of an acidic subcellular compartment in the remodelling pathway of disaturated phosphatidylcholine synthesis by granular pneumocytes.     

Dietary conjugated linoleic acid alters long chain polyunsaturated fatty acid metabolism in brain and liver of neonatal pigs

Effects of dietary conjugated linoleic acid (CLA, 1% mixed isomers) on n-6 long-chain polyunsaturated fatty acid (LCPUFA) oxidation and biosynthesis were investigated in liver and brain tissues of neonatal piglets. Fatty acid β-oxidation was measured in tissue homogenates using [1-¹⁴C]linoleic acid (LA) and -arachidonic acid (ARA) substrates, while fatty acid desaturation and elongation were traced using [U-¹³C]LA and GC-MS. Dietary CLA had no effect on fatty acid β-oxidation, but significantly decreased n-6 LCPUFA biosynthesis by inhibition of LA elongation and desaturation. Differences were noted between our ¹³C tracer assessment of desaturation/elongation and simple precursor-product indices computed from fatty acid composition data, indicating that caution should be exercised when employing the later. The inhibitory effects of CLA on elongation/desaturation were more pronounced in pigs fed a low fat diet (3% fat) than a high fat diet (25% fat). Direct elongation of linoleic acid to C20:2n-6 via the alternate elongation pathway might play an important role in n-6 LCPUFA synthesis because more than 40% of the synthetic products of [U-¹³C]LA accumulated in [¹³C]20:2n-6. Overall, the data show that dietary CLA shifted the distribution of the synthetic products of [U-¹³C]LA between elongation and desaturation in liver and decreased the total synthetic products of [U-¹³C]LA in brain by inhibiting LA elongation to C20:2n-6. The impact of CLA on brain LCPUFA metabolism of the developing neonate merits consideration and further investigation.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

The oxidation and utilization of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism, and the role of plasma phospholipids and neutral lipids in milk-fat synthesis

1. Measurements were made of milk yield, mammary blood flow and arteriovenous differences of each plasma lipid fraction, and their specific radioactivities, during the infusion of [U-¹⁴C]stearate, [U-¹⁴C]oleate, [U-¹⁴C]palmitate and [1-¹⁴C]acetate into fed lactating goats. 2. Entry rates of fatty acids into the circulation were 4·2mg./min./kg. body wt. for acetate, and 0·18, 0·28 and 0·42mg./min./kg. for stearate, oleate and palmitate respectively. Acetate accounted for 23% of the total carbon dioxide produced by the whole animal, and contributed to the oxidative metabolism of the mammary gland to about the same extent. Corresponding values for each of the long-chain acids were less than 1%. 3. There were no significant arteriovenous differences of phospholipids, sterols or sterol esters, and their fatty acid composition showed no net changes during passage through the mammary gland. 4. There were large arteriovenous differences of plasma triglycerides, and their fatty acid composition showed marked changes across the gland. The proportions of palmitate and stearate fell, and that of oleate increased. 5. Arteriovenous differences of plasma free fatty acids (FFA) were small and variable, but a large fall in the specific radioactivity of each of the long-chain acids examined indicated substantial uptake of plasma FFA, accompanied by roughly equivalent FFA release from mammary tissue. The uptake of FFA was confirmed by the extensive transfer of radioactivity into milk. The FFA of milk were similar in composition and radioactivity to the milk triglyceride fatty acids, and quite unlike plasma FFA. 6. The formation of large amounts of oleic acid (18–21 mg./min.) from stearic acid was demonstrated. 7. During the terminal stages of the [¹⁴C]acetate infusion, milk triglyceride fatty acids of chain length C₄–C₁₄ showed specific radioactivities that were 75–90% of that of blood acetate, and that of palmitate was roughly one-quarter of this value. Oleate and stearate were unlabelled. 8. The results confirmed that milk fatty acids of chain length C₄–C₁₄ arise largely from blood acetate, and palmitate is derived partly from acetate and partly from plasma triglyceride, the latter fraction being almost the sole precursor of oleate and stearate.     

The fuel of respiration of rat kidney cortex

1. In kidney-cortex slices from the well-fed rat, glucose (5mm) supplied 25–30% of the respiratory fuel; in the starved state, the corresponding value was 10%. These results are based on measurements of the net uptake of glucose and of the specific radioactivity of labelled carbon dioxide formed in the presence of [U-¹⁴C]-glucose. 2. Added acetoacetate (5mm) or butyrate (10mm) provided up to 80%, and added oleate (2mm) up to 50% of the fuel of respiration. The oxidation of endogenous substrates was suppressed correspondingly. 3. More [U-¹⁴C]oleate was removed by the tissue than could be oxidized by the amount of oxygen taken up; less than 25% of the oleate removed was converted into respiratory carbon dioxide and about two-thirds was incorporated into the tissue lipids. The rate of oleate incorporation into the neutral-lipid fraction was calculated to be equivalent to the rate of oxidation of endogenous fat, which provided the chief remaining fuel. 4. The contribution of endogenous substrates to the respiration (50%) in the presence of added oleate is taken to reflect either a high turnover rate of the endogenous neutral lipids (approx. half-life 2·5hr.) or a raised rate of lipolysis caused by the experimental conditions in vitro. 5. Added l-α-glycerophosphate (2·5mm) increased oleate incorporation into the neutral-lipid fraction by up to 40% (i.e. caused a net synthesis of triglyceride). 6. Lactate (2·5mm) added as sole substrate supplied 30% of the respiratory fuel, but with added oleate (2mm) lactate was converted quantitatively into glucose. Oleate stimulated the rate of gluconeogenesis from lactate by 45%. 7. The oxidation of both long-chain and short-chain even-numbered fatty acids was accompanied by ketone-body formation. Ketone-body synthesis from oleate, but not from butyrate, increased six- to seven-fold after 48hr. of starvation. The maximum rates of renal ketogenesis (80μmoles/hr./g. dry wt., with butyrate) were about 20% of the maximum rates observed in the liver (on a weight-for-weight basis) and accounted for, at most, 35% of the fatty acid removed. 8. dl-Carnitine (1·0mm) had no effect on the rates of uptake of acetate, butyrate or oleate or on the rate of radioactive carbon dioxide formation from [U-¹⁴C]oleate, but increased ketone-body formation from oleate by more than 100%. Ketone-body formation from butyrate was not increased. 9. There is evidence supporting the assumption that there are cells in which gluconeogenesis and ketogenesis occur together, characterized by equal labelling of [U-¹⁴C]oleate and the ketone bodies formed, and other cells that oxidize fat and do not form ketone bodies. 10. Inhibitory effects of unlabelled acetoacetate on the oxidation of [1-¹⁴C]butyrate and of unlabelled butyrate on [4-¹⁴C]acetoacetate oxidation show that fatty acids and ketone bodies compete as fuels on the basis of their relative concentrations. 11. The pathway of ketogenesis in renal cortex must differ from that of the liver, as β-hydroxy-β-methylglutaryl-CoA synthetase is virtually absent from the kidney. In contrast with the liver the kidney possesses 3-oxo acid CoA-transferase (EC 2.8.3.5), and the ready reversibility of this reaction and that of thiolase (EC 2.3.1.9) provide a mechanism for ketone-body formation from acetyl-CoA. This mechanism may apply to extrahepatic tissues generally, with the possible exception of the epithelium of the rumen and intestines.     

Studies on lipogenesis in vivo. Effect of dietary fat or starvation on conversion of [14C]glucose into fat and turnover of newly synthesized fat

1. Lipogenesis was studied in vivo by giving mice 250mg. meals of [U-(14)C]glucose and measuring the disposition and incorporation of label. About 48% of the (14)C dose was eliminated as (14)CO(2) in the first 2hr. At 60min. after administration, 1.0, 1.9 and 11.9% of the dose was recovered as liver glycogen, liver fatty acid and carcass fatty acid respectively. Of the [(14)C]glucose converted into fat in the epididymal pads about 90% was present as glyceride fatty acid and 10% as glyceride glycerol. 2. Hepatic synthesis of fatty acid was depressed by dietary fat to a much greater extent than was synthesis outside the liver. Both feeding with fat and starvation decreased the proportion of the label taken up by adipose tissue present as fat (triglyceride) and increased the proportion of triglyceride label present as glyceride glycerol. These results are consistent with the hypothesis that the primary action of both these conditions in decreasing fat synthesis is to inhibit synthesis of fatty acids. 3. Turnover of body fat labelled in vivo from [U-(14)C]glucose was estimated from the decline in radioactivity measured over the first 24hr. of the experiment. The half-life of liver and extrahepatic fatty acids (excluding epididymal fat) was 16hr. and 3 days respectively. In contrast, no measurable decrease in radioactivity of the fatty acids of epididymal fat was observed for 7 days after administration of the [U-(14)C]glucose.     

A comparison of extrahepatic lipogenesis from a small glucose meal in obob and gold thioglucose obese mice fed low- or high-fat diets with or without the addition of Delta22-5beta-taurocholenic acid

Extrahepatic fatty acid synthesis from a 250 mg meal of [U-(14)C]-glucose was measured in epidymal fat pads and the remaining carcass of hyperglycemic obese (obob), gold thioglucose obese, and nonobese controls under conditions of maximum and minimum lipogenesis. Also assessed was the effect of Delta(22)-5beta-taurocholenic acid, previously shown to inhibit hepatic fatty acid synthesis. Both types of obese and nonobese mice were fed for 6 weeks glucose-based diets containing either 1% corn oil or 40% lard with or without the addition of 0.05% taurocholenic acid. In mice fed 1% corn oil, incorporation of labeled glucose into carcass fatty acids was 25% greater in nonobese than obese mice of either type of obesity. On this diet incorporation of labeled glucose into epididymal fatty acids was reduced 83% in hyperglycemic obese mice compared with nonobese littermates. The corresponding reduction in lipogenesis in gold thioglucose obese mice was only 23% compared with nonobese controls. Feeding 40% lard reduced incorporation of labeled glucose into epididymal and carcass fatty acid 67 to 95% compared with mice fed 1% corn oil in both types of obese and nonobese mice whether or not taurocholenic acid had been fed. Both types of obesity or feeding 40% lard reduced lipogenesis in fat pads to a greater extent than glucose uptake by the pads with the reductions additive. Feeding taurocholenic acid reduced pad weight 30% across strain and obesity status, increased uptake of labeled glucose into epididymal fat pads and increased the percentage of the labeled glucose in the pad recovered as fatty acid in both types of obese and nonobese mice when the diet was 1% corn oil. Similarities and differences between the two obesity models are discussed.     

Studies on lipogenesis in vivo. Lipogenesis during extended periods of re-feeding after starvation

1. Lipogenesis was studied in mice re-fed for up to 21 days after starvation. At appropriate times [U-(14)]glucose was given by stomach tube and incorporation of (14)C into various lipid fractions measured. 2. In mice starved for 48hr. and then re-fed for 4 days with a diet containing 1% of corn oil, incorporation of (14)C from [U-(14)C]glucose into liver fatty acids and cholesterol was respectively threefold and eightfold higher than in controls fed ad libitum. The percentages by weight of fatty acids and cholesterol in the liver also increased and reached peaks after 7 days. Both the radioactivity and weights of the fractions returned to control values after 10-14 days' re-feeding. These changes could be diminished by re-feeding the mice with a diet containing 20% of corn oil. Incorporation of (14)C from [U-(14)C]glucose into extrahepatic fatty acids (excluding those of the epididymal fat pads) was not elevated during re-feeding with a diet containing either 1% or 20% of corn oil. However, incorporation of (14)C from [U-(14)C]glucose into the fatty acids of the epididymal fat pads was increased in mice re-fed with either diet, as compared with non-starved controls. 3. Lipogenesis was also studied in mice alternately fed and starved. Mice given a diet containing 1% of corn oil for 6hr./day for 4 weeks lost weight initially and never attained the weight or carcass fat content of controls fed ad libitum. Incorporation of (14)C from dietary [U-(14)C]-glucose into the fatty acids of the epididymal fat pads was elevated threefold in the mice allowed limited access to food, although the incorporation into the remainder of the extrahepatic fatty acids was not different from that found for controls. Mice given a diet containing 20% of corn oil for 6hr./day adapted to the limited feeding regimen quicker and in 4 weeks did attain the weight and carcass fat content of controls. Incorporation of (14)C from [U-(14)C]glucose into the fatty acids of the epididymal fat pads and the remainder of the extrahepatic fatty acids was respectively fivefold and threefold higher than in controls fed ad libitum. 4. The elevation in liver lipogenesis during re-feeding was greatest on a diet containing 1% of corn oil, whereas in extrahepatic tissues the increase in lipogenesis was greater when the mice were re-fed or were allowed limited access to a diet containing 20% of corn oil. These results suggest that the causes of the increased rate of incorporation of (14)C from [U-(14)C]glucose into fatty acids during re-feeding may be different in liver from that in extrahepatic tissues.     

The tissue content and turnover rates of intermediates in the biosynthesis of glycosaminoglycans in young rat skin

1. The tissue contents of hexose monophosphate, N-acetylglucosamine 6-phosphate, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-glucuronic acid were determined in the skin of young rats less than 1 day post partum. Tissue-space determinations were used to calculate their average cellular concentrations. 2. The incorporation of [U-¹⁴C]-glucose into the intermediates was recorded with time and their rates of turnover were calculated. The results demonstrated product–precursor relationships along the pathway of hexosamine synthesis and that of hexuronic acid synthesis. The rates of synthesis of UDP-N-acetylhexosamine and UDP-glucuronic acid were 1·5±0·3 and 0·24±0·03mμmoles/min./g. of tissue respectively. These results indicated the average turnover time of the total tissue glycosaminoglycans to be about 5 days.     

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.     

Quantifying conversion of linoleic to arachidonic and other n-6 polyunsaturated fatty acids in unanesthetized rats

Isotope feeding studies report a wide range of conversion fractions of dietary shorter-chain polyunsaturated fatty acids (PUFAs) to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids. In this study, whole-body (largely liver) steady-state conversion coefficients and rates of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and other elongated n-6 PUFAs were quantified directly using operational equations, in unanesthetized adult rats on a high-DHA but AA-free diet, using 2 h of intravenous [U-¹³C]LA infusion. Unesterified LA was converted to esterified LA in plasma at a greater rate than to esterified γ-linolenic (γ-LNA, 18:3n-6), eicosatrienoic acid (ETA, 20:3n-6), or AA. The steady-state whole-body synthesis-secretion (conversion) coefficient to AA equaled 5.4 × 10⁻³ min⁻¹, while the conversion rate (coefficient × concentration) equaled 16.1 μmol/day. This rate exceeds the reported brain AA consumption rate by 27-fold. As brain and heart cannot synthesize significant AA from circulating LA, liver synthesis is necessary to maintain their homeostatic AA concentrations in the absence of dietary AA. The heavy-isotope intravenous infusion method could be used to quantify steady-state liver synthesis-secretion of AA from LA under different conditions in rodents and in humans.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

Studies on lipogenesis in vivo. Effects of starvation and re-feeding, and studies on cholesterol synthesis

1. Studies in vivo have been carried out on hepatic and extrahepatic cholesterol synthesis and also on the effects of starvation and re-feeding on both cholesterol and fatty acid synthesis. 2. In rats and mice fed on a stock diet, extrahepatic tissues accounted for about 4 times as much newly synthesized cholesterol as did the liver. The liver appeared to be somewhat more important in the rat than the mouse. Feeding with cholesterol greatly decreased and cholestyramine greatly increased hepatic cholesterol synthesis without much effect on extrahepatic synthesis. 3. Mice starved for up to 7hr. did not lose any of the ability to convert a [U-(14)C]glucose meal into fat, whereas 18hr. of starvation resulted in an 80% loss of fatty acid synthesis in liver and carcass, an 80% loss in liver cholesterol synthesis and a 65% decrease in carcass cholesterol synthesis; 18hr. of food deprivation also decreased the proportion of counts in epididymal fat pads present as fat and increased the proportion present as glyceride glycerol. 4. Re-feeding for up to 7hr. restored fatty acid synthesis from a [U-(14)C]glucose meal to about 50% of the values for non-starved mice but had no effect on hepatic cholesterol synthesis. The altered distribution of counts in the epididymal fat pads caused by starvation was restored to normal after feeding for 1hr.     

Effect of triperidol on carbohydrate and amino acid metabolism in rat brain-cortex slices

1. The effect of triperidol on the metabolism of glucose, pyruvate, glutamate, aspartate and glycine was studied with rat brain-cortex slices, U-¹⁴C-labelled substrates and a quantitative radiochromatographic technique. 2. Triperidol at a concentration of 0·2mm decreased the oxygen uptake and the ¹⁴CO₂ production by about 30% when glucose, pyruvate and glutamate were used as substrates, whereas no effects were observed with aspartate and glycine. 3. The drug did not alter qualitatively the metabolic pattern of the substrates. 4. Quantitatively, triperidol decreased the incorporation of ¹⁴C from [U-¹⁴C]glucose and [U¹⁴-C]-pyruvate into glutamate, glutamine and γ-aminobutyrate but not into lactate, alanine and aspartate. The overall utilization rates of glucose and pyruvate were decreased. The relative specific radioactivities of glutamate and aspartate were also decreased. 5. Triperidol increased the rate of disappearance of U-¹⁴C-labelled glutamate, aspartate and glycine from the incubation medium, and altered the distribution of their metabolites between medium and tissue. 6. No appreciable effect of triperidol on [1-¹⁴C]galactose disappearance was found.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of palmitate and lipolytic agents

1. 0.5mm-Palmitate stimulated incorporation of [U-¹⁴C]glucose into glyceride glycerol and fatty acids in normal fat cells in a manner dependent upon the glucose concentration. 2. In the presence of insulin the incorporation of 5mm-glucose into glyceride fatty acids was increased by concentrations of palmitate, adrenaline and 6-N-2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate up to 0.5mm, 0.5μm and 0.5mm respectively. Higher concentrations of these agents produced progressive decreases in the rate of glucose incorporation into fatty acids. 3. The effects of palmitate and lipolytic agents upon the measured parameters of glucose utilization were similar, suggesting that the effects of lipolytic agents are mediated through increased concentrations of free fatty acids. 4. In fat cells from 24h-starved rats, maximal stimulation of glucose incorporation into fatty acids was achieved with 0.25mm-palmitate. Higher concentrations of palmitate were inhibitory. In fat cells from 72h-starved rats, palmitate only stimulated glucose incorporation into fatty acids at high concentrations of palmitate (1mm and above). 5. The ability of fat cells to incorporate glucose into glyceride glycerol in the presence of palmitate decreased with increasing periods of starvation. 6. It is suggested that low concentrations of free fatty acids stimulate fatty acid synthesis from glucose by increasing the utilization of ATP and cytoplasmic NADH for esterification of these free fatty acids. When esterification of free fatty acids does not keep pace with their provision, inhibition of fatty acid synthesis occurs. Provision of free fatty acids far in excess of the esterification capacity of the cells leads to uncoupling of oxidative phosphorylation and a secondary stimulation of fatty acid synthesis from glucose.     

Determination of C distribution in photosynthetic serine and phosphoglycerate from grape leaves

Serine and phosphoglyceric acid are the classical marker intermediates of photorespiration and reductive carbon assimilation in C₃ plants. The present paper introduces a new and fast method for the determination of ¹⁴C distribution in these compounds by selective elimination of C-3 (NaIO₄) or C-1 (ninhydrin/ceric sulfate). Reproducibility of the procedure was found to be better than ±1% upon degradation of [U-¹⁴C]serine and [U-¹⁴C]glycerate standards.