Cellular energy metabolism during fetal development. II. Fatty acid oxidation by the developing heart

In view of the importance of fatty acids as substrates for the mature heart, fatty acid oxidation by fetal and calf heart mitochondria has been investigated. Free fatty acids of 10 carbon units or less which exhibit carnitine-independent transport into mitochondria were effective substrates for oxidative phosphorylation in both fetal and calf heart mitochondria. Efficient oxidative phosphorylation with these substrates was dependent upon the presence of bovine serum albumin in the assay medium to reverse the uncoupling effects of the fatty acids. In the presence of bovine serum albumin, ADP/0 ratios were in the range of 3 when short-chain fatty acids and carnitine esters of short- and long-chain fatty acids were substrates. Compared with calf heart mitochondria, fetal heart mitochondria showed decreased carnitine-dependent oxidation of palmityl-CoA. However, the oxidation of palmitylcarnitine was identical in both. These data suggest that the formation of palmitylcarnitine is rate limiting for palmityl-CoA oxidation by the fetal heart mitochondria and that long-chain fatty acids are not readily oxidized by the fetal heart.     

Cellular energy metabolism during fetal development. IV. Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.     

Fatty acid synthesis by slices from developing leaves

Fatty acid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivium L.) leaves. The basal regions of the leaves had the lowest rates of fatty acid synthesis and accumulated small amounts of very long chain fatty acids. Fatty acid synthesis was highest in the middle leaf sections in all four plants. Linolenic acid synthesis from [1-¹⁴C]acetate was highest in the distal leaf sections of rye grass. The labelling of the fatty acids of individual lipids of rye grass was examined and it was found that [¹⁴C]linolenic acid was highest in the galactolipids. Synthesis of this acid in the galactolipids was most active in leaf segment C. Only traces of [¹⁴C]linolenic acid were ever found in phosphatidylcholine and it is concluded that this phospholipid cannot serve as a substrate for linoleic acid desaturation in rye grass. The synthesis of fatty acids was sensitive to arsenite, fluoride and the herbicide EPTC. The latter was only inhibitory towards those leaf segments which made very long chain fatty acids. Formation of fatty acids from [1-¹⁴C]acetate was also studied in chloroplasts prepared from successive leaf sections of rye grass. Chloroplasts isolated from the middle leaf sections had the highest activity. Palmitic and oleic acids were the main fatty acid products in all chloroplast preparations. Linolenic acid synthesis was highest in chlorplasts isolated from the distal leaf sections of rye grass.     

Fatty acid synthesis in fetal lung

De novo fatty acid synthesis in lung is significant during fetal growth and development. Specific activity and relative rate of synthesis of fatty acid synthetase increase with the days of gestational age and drop significantly after birth. Fetal lungs contain thyroid hormone receptors and binding capacities of this hormone to the fetal lungs also increase with the days of gestational age. Our results suggest that de novo fatty acid synthesis in fetal lungs may make a significant contribution towards surfactant synthesis.     

Fatty acid metabolism in Paramecium. Oleic acid metabolism and inhibition of polyunsaturated fatty acid synthesis by triparanol

Paramecium requires oleic acid for growth and can grow in media containing no other fatty acids. In the present study, we have shown that this ciliate utilized oleate mainly as a carbon and energy source, even though this fatty acid was the only substrate available for synthesis of polyunsaturated fatty acids. Culture growth was inhibited by the addition of the drug triparanol. Triparanol decreased the formation of polyunsaturated fatty acids from oleate by preventing desaturation to form the dienoic acid, linoleate. Triparanol inhibition resulted in an altered phospholipid fatty acyl composition, an increased fragility and an altered behavioral response of the cells to a depolarizing stimulation solution. Therefore, although most of the dietary oleate was not used by the cells for polyunsaturated fatty acid synthesis, the desaturation of oleic acid was critical for normal culture growth, cell integrity and swimming behavior, all of which are expected to be dependent on normal membrane lipid composition.     

Fatty acid and lactate metabolism by heart homogenates from alloxan-diabetic dogs

Myocardial homogenates from control of alloxan-diabetic dogs were incubated in the presence of lactate or palmitate as the sole substrate. The rate of lactate oxidation of the homogenates from diabetic dogs was less than half of the oxidation rate of homogenates from control animals. The incorporation of labelled palmitate into triglyceride was greatly enhanced in the heart taken from diabetic animals. The tissue concentration of l-carnitine was twice and that of triglyceride was three times as high in the diabetic animals as in their control counterparts. Thus the myocardium of diabetic animals exhibited both altered substrate utilization and increased rate of triglyceride synthesis.     

Fatty acid composition and lipid synthesis in developing safflower seeds

Linoleic acid predominated in every lipid class during the whole period of seed development of safflower, while linolenic acid decreased with increasing maturation and it was not detected in mature seeds. Just before the initiation of triacylglycerol accumulation, the fatty acid composition of triacylglycerols changed more rapidly than those of phospholipids and glycolipids. Saturated fatty acids tended to accumulate at the 1- and 3-positions of the glycerol molecule and the more highly unsaturated acids at the 2-position. The fatty acid compositions at the 1- and 3-positions were similar in all cases investigated, but in none of the triacylglycerols was the distribution completely symmetrical. The positional distribution of linolenic acid in triacylglycerols prepared from the immature seeds 2 days after flowering and from the leaves was unusual; in spite of its highest degree of unsaturation, it was preferentially esterified at the 1- and 3-positions. When triacylglycerol was most rapidly accumulated (14–18 days after flowering), the incorporation of acetate-[U- ¹⁴C] into total lipids was also maximum and dienoic fatty acids were the principal acids labelled. Diacylglycerols and compound lipids reached the highest rate of synthesis 15 days after flowering, and then a maximum incorporation into triacylglycerol occurred 18 days after flowering. Incubation temperature affected the synthesis of individual lipid classes. Triacylglycerol was more rapidly synthesized at 32° than at 10°, while diacylglycerols and compound lipids were accumulated under the low-temperature condition. A rise of incubation temperature caused a depression in dienoic acid synthesis.     

Fatty acid synthesis in the fetal lung: relationship to surfactant lipids

The aims of this study were to investigate the control of fatty acid synthesis and its relationship to surfactant production in the fetal lung during alteration of hormonal and substrate conditions. Lung explants from 18 day fetuses (term = 22 days) which were cultured 2 days in the presence of 10 mM lactate showed parallel acceleration of de novo fatty acid synthesis (3H2O incorporation) and [14C]choline incorporation into disaturated phosphatidylcholine (DSPC) compared to culture of explants in glucose. Both the cultured and fresh explants were resistant to the classical short term (4 h) cAMP inhibition of fatty acid synthesis with 3 mM dibutyryl cAMP or 0.5 mM aminophylline. In the cultured explants short term cAMP elevation increased DSPC production, and long term (2 day) cAMP elevation caused a further increase in DSPC synthesis and also stimulated fatty acid synthesis. In cultured explants from 17 day fetuses, dexamethasone (0.1 microM) caused a synergistic increase with aminophylline in both fatty acid synthesis and DSPC production whereas, in explants from 18 day fetuses, dexamethasone inhibited both processes and reduced the level of stimulation of DSPC and fatty acid synthesis seen with aminophylline alone. Dexamethasone also reduced the stimulation of both DSPC and fatty acid synthesis produced in the culture of 18 day explants with bacitracin (0.5 mg/ml), whereas the combination of bacitracin and aminophylline resulted in a synergistic increase in DSPC production. Culture with glucagon (0.1 microM) also stimulated DSPC synthesis but at physiological levels insulin had no effect on either DSPC or fatty acid synthesis. These data show that lung fatty acid synthesis exhibits unique features of fatty acid synthesis regulation compared to other lipogenic tissues and also suggest a link between de novo fatty acid synthesis and surfactant production during the critical period of accelerated lung maturation.     

Cerebral energy metabolism in guinea pig fetuses during development.

During development fetal arterial oxygen tension falls, whereas cerebral oxygen consumption rises due to an increase in cerebral metabolism. To compensate for this increase in oxygen consumption, blood flow and therefore oxygen delivery to the cerebrum rises. To determine whether during development oxygen delivery to the cerebrum meets cerebral oxygen consumption, we measured the concentrations of high-energy phosphates and glycolytic intermediates in the cerebral cortex of fetal guinea pigs at different gestational ages. During development there was no change in the concentrations of adenosine triphosphate, creatine phosphate, adenosine monophosphate, and lactate. However, cerebral concentrations of adenosine diphosphate increased and those of glucose decreased. Our results suggest that the increase in fetal cerebral oxygen delivery during development meets cerebral oxygen consumption with increasing gestational age. We speculate that the measured rise in the concentrations of adenosine diphosphate may accelerate glycolysis during development and therefore may cause a rise in both cerebral blood flow to maintain oxygen delivery.     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Hepatic bile acid metabolism during early development revealed from the analysis of human fetal gallbladder bile

A detailed study of the qualitative and quantitative composition of bile acids in human fetal gallbladder bile is described. Bile was collected during early gestation (weeks 16-19) and analyzed by gas chromatography and mass spectrometry, fast atom bombardment ionization mass spectrometry, and high performance liquid chromatography. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex LH-20. Quantitatively more than 80% of the bile acids were secreted into bile conjugated to taurine. Unconjugated bile acids and glycine conjugates accounted for 5-10% of the total biliary bile acids. Bile acid sulfates were present only in trace amounts indicating that quantitatively sulfation is not an important pathway in bile acid metabolism during development. Total biliary bile acid concentrations were low (0.1-0.4 mM) when compared to reported values for adult bile (greater than 10 mM). Chenodeoxycholic acid was the major biliary bile acid and exceeded cholic acid concentrations by 1.43-fold indicating either a relative immaturity in 12 alpha-hydroxylase activity during early life or a dominance of alternative pathways for chenodeoxycholic acid synthesis. A relatively large proportion of the biliary bile acids comprised metabolites not found in adult bile. The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1 and C-6 hydroxylation are important pathways in bile acid synthesis during development. We describe, for the first time, evidence for the existence of a C-4 hydroxylation pathway in the metabolism of bile acids, which may be unique to early human development. Mass spectrometry was used to confirm the identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic and 3 alpha,4 beta-dihydroxy-5 beta-cholanoic acids. Quantitatively, these C-4 hydroxylated bile acids accounted for 5-15% of the total biliary bile acids of the fetus, suggesting that C-4 hydroxylation is quantitatively an important pathway in the bile acid metabolism during early life.     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.