Synthesis Of Medium-chain Fatty Acids Research Articles

Synthesis of medium-chain fatty acids and their incorporation into triacylglycerols by cell-free fractions fromCuphea embryos

Synthesis of medium-chain fatty acids and their incorporation into triacylglycerols by cell-free fractions fromCuphea embryos

Synthesis of medium-chain fatty acids and their incorporation into triacylglycerols by cell-free fractions from Cuphea embryos

During their rapid maturation period, seeds of Cuphea wrightii A. Gray mainly accumulate medium-chain fatty acids (C8 to C14) in their storage lipids. The rate of lipid deposition (40-50 mg·d(-1)·(g fresh weight)(-1)) is fourfold higher than in seeds of Cuphea racemosa (L. f.) Spreng, which accumulate long-chain fatty acids (C16 to C18). Measurements of the key enzymes of fatty-acid synthesis in cell-free extracts of seeds of different maturities from Cuphea wrightii show that malonyl-CoA synthesis may be a triggering factor for the observed high capacity for fatty-acid synthesis. Experiments on the incorporation of [1-(14)C]acetate into fatty acids by purified plastid preparations from embryos of Cuphea wrightii have demonstrated that the biosynthesis of medium-chain fatty acids (C8 to C14) is localized in the plastid. Thus, in the presence of cofactors for lipid synthesis (ATP, NADPH, NADH, acyl carrier protein, and sn-glycerol-3-phosphate), purified plastid fractions predominantly synthesized free fatty acids, 30% of which were of medium chain length. Transesterification of the freshly synthesized fatty acids to coenzyme A and recombination with the microsomal fraction of the embryo homogenate induced triacylglycerol synthesis. It also stimulated fatty-acid synthesis by a factor 2-3 and increased the relative amount of medium-chain fatty acids bound to triacylglycerols, which corresponded to about 60-80% in this lipid fraction.

Medium-chain fatty acid synthesis.

Medium-chain fatty acid synthesis.

Fatty acid synthesis by explant cultures from the mammary glands of goats on days 60 and 120 of pregnancy.

Explants of mammary glands from 60-day pregnant goats showed a mean fourfold increase in fatty acid synthesis from acetate when cultured with insulin+ cortisol. Epithelial cells increased their area by 60% but no secretory activity was induced. In 120-day pregnant goats, fatty acid synthesis and epithelial cell area were greater than at day 60 of pregnancy and were unaffected by hypophysectomy or by daily treatment with bromocriptine from day 60. Neither increased further on culture of mammary explants in insulin + cortisol. Ovine prolactin increased fatty acid synthesis two-fold when added to insulin + cortisol in cultures of mammary tissue from goats on day 60 of pregnancy and secretory activity was induced. On day 120 of pregnancy insulin + cortisol + prolactin sustained or slightly stimulated both fatty acid synthesis and the extensive secretion present in the tissue at the start of culture. Synthesis of medium-chain fatty acids of milk-fat was also sustained by prolactin in one goat. An atmosphere of air was found to maintain normal histological structure of the mammary gland. By contrast, in 95% oxygen, explants from goats which were 60 days pregnant showed epithelial cells filling the lumina of ducts and alveoli in 60% of explants and a poor response to prolactin.

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.

Localization of thioesterase II, the chain-length regulatory enzyme of milk fatty acid synthesis, in rat mammary gland epithelial cells.

Two approaches were used to establish the intercellular distribution of fatty acid synthetase and thioesterase II in the lactating rat mammary gland. Thioesterase II is the chain-length regulatory enzyme in the biosynthesis of the medium-chain fatty acids characteristic of milk fat. Using immunohistochemical techniques, immunoreactive fatty acid synthetase was found in both mammary adipocytes and epithelial cells; in contrast, immunoreactive epithelial cells were isolated from lactating rat mammary glands after digestion with collagenase and thermolysin, and their lipogenic activity was studied using isotopically labelled acetate. Consistent with the immunohistochemical data, adipocytes synthesized exclusively long-chain fatty acids whereas epithelial cells synthesized predominantly chain fatty acids. The results indicate that the capacity for synthesis of medium-chain fatty acids is a unique property of the epithelial cell component of the mammary gland.

Development of the capacity of mouse mammary glands for medium chain fatty acid synthesis during pregnancy and lactation

The time-course for appearance of fatty acid synthetase and thioesterase II, enzymes required for the synthesis of medium chain (C 8-C 12) fatty acids by the mammary gland, has been studied in the mouse and compared with that in the rat. The development of high levels of fatty acid synthetase in the mouse mammary gland during the early days of lactation coincided with an observed increase in the overall lipogenic capacity of the gland, assessed by measuring the incorporation of radioactive acetate into fat in tissue slices. Both the level of thioesterase II in the gland and the proportion of medium chain fatty acids synthesized from acetate by tissue slices began to increase in late gestation but did not reach a maximum until lactation was well established. In this regard the mouse is distinctly different from the rat, which establishes maximum levels of thioesterase II in the mammary gland prior to parturition. The significance of this difference as it relates to mammary gland development is discussed.

Hormonal regulation of medium chain fatty acid synthesis by mouse mammary gland explants

AbstractExplants of pregnant mouse mammary tissue were cultured in media supplemented with various hormones. During the last few hours of culture, explants were labeled with [14C] acetate. Fatty acid synthesis by the tissue was analyzed using reverse phase thin layer chromatography, and incorporation of radioactivity into the medium chain fatty acid (MCFA) fraction was calculated as total MCFA per mg tissue fresh weight and as a percentage of the total fatty acid radioactivity (%MCFA). After 48 hr of culture, explants had an elevated %MCFA synthesis only when exposed to media containing insulin, cortisol and prolactin, confirming previous reports. Increasing doses of prolactin (maximal response at 300 mg/ml) caused a coordinate rise in both %MCFA synthesis and total MCFA synthesis per mg fresh weight. Epithelial cells isolated from explants after culture for 48 hr with insulin, cortisol and prolactin showed an elevated %MCFA synthesis compared to cells from explants cultured with insulin and cortisol alone, indicating that hormonal stimulation of mammary explants causes a change in the chain length of fatty acids synthesized by mammary epithelial cells. The specificity of the explant response to prolactin was tested with other hormones: FSH and calcitonin had no effect, whereas bovine growth hormone showed activity only at high concentration. Progesterone and 17β‐estradiol also had no effect. The analysis of MCFA synthesis provides another means of (a) assessing hormonal action upon mammary tissue, and (b) evaluating the biological activity of prolactin.

Asynchronous appearance of two enzymes concerned with medium chain fatty acid synthesis in developing rat mammary gland.

Asynchronous appearance of two enzymes concerned with medium chain fatty acid synthesis in developing rat mammary gland.

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue.

Fatty‐Acid Synthesis in Lactating‐Goat Mammary Gland 1. Medium‐Chain Fatty‐Acid Synthesis

Tissue slices from lactating goat-mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long-chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle-free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short-chain and long-chain fatty acids. Terminating acyl-thioesterases of low molecular weight could not be detected in the particle-free supernatant. Addition of the microsomal fraction to the particle-free supernatant induced the synthesis of medium-chain fatty acids in proportions which were similar to those found in goat milk fat.

Fatty‐Acid Synthesis in Lactating‐Goat Mammary Gland 2. Medium‐Chain Fatty Acid Synthesis

Addition of goat, rat, rabbit and cow mammary microsomal fraction to fatty acid synthetase of goat mammary gland induced C10 fatty acid synthesis. When the microsomal fractions from rat, rabbit and cow mammary gland were incubated with their respective fatty acid synthetases only the cow enzyme synthesized significant amounts of C10 compared to the purified fatty acid synthetases alone. The goat mammary microsomal fraction was not able to induce C10 synthesis by rat and rabbit mammary-gland fatty acid synthetase, but only by the goat and cow enzyme. Both goat and cow mammary-gland fatty acid synthetase have in contrast to the rabbit and rat enzymes the ability to hydrolyse medium-chain acyl-thioesters. We therefore suggest a novel mechanism for medium-chain fatty acid synthesis in lactating-goat mammary gland. This mechanism involves an activation of the medium-chain acyl-thioester hydrolase within the fatty acid synthetase of goat mammary gland by an unknown microsomal factor.

Inhibition by low concentrations of ouabain of prolactin-induced lactogenesis in rabbit mammary-gland explants.

1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37 degrees C in air for 24-48h in Medium 199 buffered with 20mm-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na(+)/K(+)-dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K(+), and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na(+) and K(+) concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37 degrees C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K(+) in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10mum) and increased Na(+) and decreased K(+) concentrations in the tissue. 3. Ouabain at concentrations up to 1mum did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1mum-ouabain. 4. Explants cultured in the presence of 2mum-ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na(+)/K(+)-dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.

The interaction of fatty acid synthetase with cytoplasmic protein in the control of the chain-length of fatty acids synthesised by the lactating rabbit mammary gland

1. 1. Fatty acid synthetase from rabbit mammary gland can be separated from the protein which modifies the chain-length at which fatty acids are released from the enzyme complex in the soluble fraction. This can be achieved by ultracentrifugation, precipitation with specific antibody or ammonium sulphate. 2. 2. The chain-length modifying protein in the supernatant fraction from rabbit mammary gland was less active towards cow mammary gland fatty acid synthetase than rabbit mammary gland fatty acid synthetase in the synthesis of medium-chain fatty acids. The fatty acid synthetases from these two tissues are also immunologically non-identical. 3. 3. It is proposed that there is a loose but specific interaction of rabbit mammary gland fatty acid synthetase with the chain-length modifying protein in regulating product chain length which is dependent on the concentration of interacting proteins. 4. 4. The chain-lengthening effect of added malonyl-CoA decreases with increasing concentration of interacting proteins, but differences in the fatty acid chain-length with malonyl-CoA synthesised in situ by acetyl-CoA carboxylase and with added malonyl-CoA indicate that the product chain-length is sensitive to the availability of malonyl-CoA for enlongation in all but the most tightly coupled situations.

Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis

1. An acyl-thioester hydrolase was isolated from the cytosol of lactating-rabbit mammary gland. The purified enzyme terminates fatty acid synthesis at medium-chain (C8:0-C12:0) acids when it is incubated with fatty acid synthetase and rate-limiting concentrations of malonyl-CoA. These acids are characteristic products of the lactating gland. 2. The mol.wt. of the enzyme is 29000+/-500 (mean+/-S.D. of three independent preparations), as estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. The enzyme also hydrolyses acyl-CoA esters of chain lengths C10:0-C16:0 when these are used as model substrates. The greatest activity was towards dodecanoyl-CoA, and the three preparations had specific activities of 305, 1130 and 2010 nmol of dodecanoyl-CoA hydrolysed/min per mg of protein when 56muM substrate was used. 4. The way in which this enzyme controls the synthesis of medium-chain fatty acids by fatty acid synthetase is briefly discussed.

The synthesis of medium-chain fatty acids by lactating-rabbit mammary gland studied in vitro

The synthesis of medium-chain fatty acids by lactating-rabbit mammary gland studied <i>in vitro</i>

Fatty acid biosynthesis: X. Specificity for chain-termination of fatty acid biosynthesis in cell-free extracts of lactating rabbit mammary gland

1. 1. The pattern of fatty acids synthesised from [1− 14C]acetate by subcellular fractions of lactating rabbit mammary gland fortified by appropriate cofactors was found to be dependent on the concentration of the fractions as measured by protein. 2. 2. At high concentrations (14–22 mg protein/ml), the cell-free homogenate, the microsomal plus particle-free supernatant, and the particle-free supernatant fractions all synthesised a high proportion of the medium-chain fatty acids ( C 8:0) and C 10:0) which are characteristic of rabbit milk. 3. 3. Dilution of these fractions to less than 1 mg protein/ml caused a progressive increase in the proportion of long-chain acids ( C 14:0 and C 16:0) synthesised. 4. 4. With concentrated subcellular fractions, synthesis of medium-chain fatty acids was not dependent on the rate of fatty acid synthesis, the concentration of malonyl-CoA, or the amount of fatty acid synthetase present. 5. 5. Though there was a correlation between the synthesis of medium-chain fatty acids and of triglyceride by particulate fractions, esterification of synthesised fatty acids did not influence the proportion of medium-chain acids formed. 6. 6. Evidence is presented that chain termination at C 8:0 and C 10:0 acids is controlled by a factor (or factors) present in the particle-free supernatant fraction. This factor is not lost on dialysis, and can be precipitated between 40 and 100% saturation with (NH 4) 2SO 4 or by heating.

Regulation of the chain-length of fatty acids synthesized by cell-free preparations of lactating rabbit, rat and guinea-pig mammary gland

1. 1. The synthesis of medium-chain fatty acids by cell-free extracts of rabbit and rat mammary glands in proportions similar to those synthesized in vivo only occurred at high protein concentrations. The inability of subcellular fractions of guinea-pig mammary gland to synthesize medium-chain fatty acids correlates with the presence of only long-chain fatty acids in guinea-pig milk fat. 2. 2. Dilution of subcellular fractions progressively increased the proportion of long-chain fatty acids synthesized until the pattern resembled that obtained with isolated fatty acid synthetase. 3. 3. The specificity of fatty acid synthesis was not influenced by the esterification of the synthesized fatty acids into glycerides. 4. 4. Fatty acid synthetase may interact with other protein components in the particle-free supernatant fraction of rabbit and rat mammary gland, thereby altering its chain-length specificity.

The synthesis of medium-chain fatty acids by lactating-rabbit mammary gland studied in vitro.

The proportion of C(8:0) and C(10:0) fatty acids synthesized by the microsomal plus particle-free supernatant fraction from lactating rabbit mammary gland is enhanced at high protein concentrations. This fraction appears to contain a soluble high-molecular-weight factor that modifies the specificity of the fatty acid synthetase complex for termination of the growing acyl chain.

Interactions of insulin, corticosterone and prolactin in promoting milk-fat synthesis by mammary explants from pregnant rabbits.

1. The rate of fatty acid synthesis by mammary explants from rabbits pregnant for 16 days or from rabbits pseudopregnant for 11 days was stimulated up to 15-fold by culturing for 2-4 days with prolactin. This treatment initiated the predominant synthesis of C(8:0) and C(10:0) fatty acids, which are characteristic of rabbit milk. 2. Inclusion of insulin in the culture medium increased the rate of synthesis of these medium-chain fatty acids. By contrast the inclusion of corticosterone led to the predominant synthesis of long-chain fatty acids. When explants were cultured for 2-4 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased up to 42-fold, but both medium- and long-chain fatty acids were synthesized. 3. These results show that the stimulus to mammary-gland lipogenesis and the initiation of synthesis of medium-chain fatty acids observed between days 16 and 23 of pregnancy in the rabbit can be simulated in vitro by prolactin alone. 4. When mammary explants from rabbits pregnant for 23 days were cultured for 2 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased fivefold, but there was a preferential synthesis of long-chain fatty acids. Culture with prolactin alone had little effect on the rate or pattern of fatty acids synthesized. 5. The results are compared with findings in vivo on the control of lipogenesis in the rabbit mammary gland, and are contrasted with the known effects of hormones in vitro on the mammary gland of the mid-pregnant mouse.