Biochemistry of development in insects. Stereospecific incorporation of fatty acids into triacylglycerols.

1. Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time-course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn-positions of triacylglycerols has been followed. 2. Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn-1 and sn-3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn-3 position at a higher rate than it acylated the other sn-positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates. Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3). 3. Kinetics of incorporation of labelled fatty acids into the sn-positions points to a non-random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.     

Changes in lymphokine receptor expression and fatty acid composition of phospholipids and triacylglycerols in rat adipocytes associated with lymph nodes following a transient immune challenge

Single-photon counting fluorimetry was used to record the time course of the expression of interleukin-10 receptors labelled with fluorescent antibodies on the surface of adipocytes over 24h, following an immune challenge to the rat popliteal lymph node. Homologous perinodal and remote-from-node samples from the stimulated and unstimulated popliteal depots were compared in rats fed on plain chow and chow supplemented with 10% w/w suet, fish or vegetable oils. Receptor expression was maximal 6 h after stimulation, and returned to baseline after 24 h, and was similar in the stimulated and unstimulated depots. Fewer receptors were elicited in tissues from rats fed lipid-supplemented diets compared with the control diet, with fewest of all following the fish oil diet. These data suggest that interleukin-10 is involved in local interactions between perinodal adipocytes and lymph node lymphoid cells. Both triacylglycerols and phospholipids contained more polyunsaturates and fewer saturates in perinodal adipose tissue than in samples from sites not associated with lymphoid tissue. These data are consistent with paracrine interactions between perinodal adipocytes and activated lymphoid cells.     

Esterification of exogenous and endogenous fatty acids by rat adipocytes in vitro.

Rat adipocytes were used in vivo to compare the esterification of exogenous fatty acids and fatty acids formed de novo from glucose or acetate. Pure single fatty acids added to the medium were esterified at comparable rates but marked differences were observed when the same acids were supplied as components of a fatty acid mixture of a composition similar to that in the tissue. Fatty acids synthesised de novo from acetate by adipocytes in a medium containing high concentrations of acetate were located predominantly in diacylglycerols. The effect was most marked with adipocytes from older rats and was enhanced by the presence of exogenous long-chain fatty acids. Exogenous oleic acid was esterified predominantly into triacylglycerols at all concentrations of acetate. No such accumulation of endogenously-synthesised fatty acids in diacylglycerols occurred when glucose was the precursor for fatty acid synthesis. The diacylglycerols formed were almost entirely of the sn-1,2-configuration.     

Mechanism of free fatty acid re-esterification in human adipocytes in vitro

Within adipose tissue, free fatty acids liberated by lipolysis may be re-esterified into newly synthesized triacylglycerol. We hypothesized that re-esterification may occur via an extracellular route, such that free fatty acids arising from lipolysis must leave the adipocyte and be taken up again before they can be re-esterified. We simultaneously measured rates of lipolysis, acylglycerol synthesis, and free fatty acid re-esterification in human adipose tissue and isolated adipocytes in vitro, utilizing a dual-isotopic technique. We manipulated incubations to increase mixing of released free fatty acids with the incubation medium. Such manipulations should decrease the probability that released free fatty acids would be taken up and re-esterified. We found that re-esterification was decreased in isolated adipocytes compared to fragments of tissue, in shaken compared to unshaken incubations, and in low adipocyte concentrations compared to high adipocyte concentrations. Rates of acylglycerol synthesis and lipolysis were unaltered by these manipulations, indicating that changes in free fatty acid re-esterification are not secondary to effects on these processes. The results are consistent with an extracellular route for free fatty acid re-esterification. Such a mechanism suggests that adipose tissue blood flow may play an important role in the regulation of free fatty acid release from adipose tissue.     

Origin of hydrogen required for fatty acid synthesis in isolated rat adipocytes.

Beef liver and beef spinal cord d-glycerate dehydrogenases have been shown to be extremely similar. No differences between the two enzymes could be shown by polyacrylamide electrophoresis, sodium dodecyl sulfate polyacrylamide electrophoresis, immunodiffusion, immunoelectrophoresis, or their response to certain inhibitors. Differences could be obtained, however, between the beef spinal cord enzyme and the hog spinal cord enzyme by immunodiffusion and immunoelectrophoresis.Only by the very sensitive technique of microcomplement fixation could a small but significant difference be shown between the beef liver and beef spinal cord enzymes. Like the beef liver and hog spinal cord enzymes, the beef spinal cord enzyme was not inhibited by high concentrations of serine or glycine. The enzyme was inhibited however by low concentrations of phosphohydroxypyruvate and by other phosphorylated compounds.     

The short-term regulation of fatty acid synthesis in the rat epididymal adipocytes

Lipogenesis and fatty acid synthetase (FAS) activity of isolated rat adipocytes that were treated with insulin or epinephrine were studied. Insulin stimulated incorporation of radioactivity from D-[U-14C]glucose into CO2, saponifiable and non-saponifiable fractions, whereas epinephrine promoted lipolysis and oxidation of glucose into CO2. Whereas insulin stimulated fatty acid synthesis, epinephrine had no effect. Changes in FAS specific activity of insulin- or epinephrine-treated adipocytes were insignificant and could not account for insulin-stimulated lipogenesis. Rat adipocyte FAS, unlike hepatic FAS, was not subject to short-term regulation by insulin, although fatty acid synthesis showed such a response.     

不同环境条件下向日葵油及脂肪酸含量研究

试验在巴基斯坦Arid农业大学进行,于2001年春、秋两季分别调查向日葵杂交种的油和脂肪酸含量,其中5个向日葵杂交种按完全随机区组设计,春向日葵在3月播种,6月收获;秋向日葵在8月播种,11月收获。调查结果表明,油和油酸的含量,春向日葵比秋向日葵高;软脂肪酸和亚油酸含量,秋向日葵比春向日葵高。因此,依据油和油酸的含量来推断,春向日葵比秋向日葵有优势;从亚油酸角度判断,秋向日葵比春向日葵更有优势。     

Regiospecific distribution of fatty acids in triacylglycerols of Artemia franciscana nauplii enriched with fatty acid ethyl esters

This paper reports the positional distribution of fatty acids in triacylglycerols (TAG) of Artemia franciscana nauplii enriched with each of palmitic (16:0), oleic (18:1n-9), linoleic (18:2n-6), linolenic (18:3n-3), eicosapentaenoic (20:5n-3), and docosahexaenoic (22:6n-3) acid ethyl esters. TAG extracted from the enriched and unenriched nauplii were subjected to regiospecific analysis to determine the fatty acid compositions of the sn-1(3) and sn-2 positions of TAG. In the unenriched nauplii, 16:0, 18:1n-9, and 18:2n-6 were preferentially located in the sn-1(3) position followed by the sn-2 position [i.e. sn-1(3)>sn-2], whereas 18:3n-3 was concentrated in the sn-2 position [i.e. sn-2>sn-1(3)]. Contents of 20:5n-3 and 22:6n-3 were low. After the nauplii were enriched with each of the ethyl esters for 18 h, fatty acid fed to the nauplii showed higher content in the sn-1(3) position than in the sn-2 position [i.e. sn-1(3)>sn-2]. Distribution pattern of 18:3n-3 changed from sn-2>sn-1(3) to sn-1(3)>sn-2 during the enrichment with 18:3n-3 ethyl ester. Increases in all of the fatty acids in TAG were attributed to that in the sn-1(3) position much more than that in the sn-2 position. Artemia nauplii appear to be characterized by preferential incorporation of exogenous fatty acids into the sn-1(3) position of TAG, even though endogenous fatty acids are esterified in the opposite position.     

Stoichiometry of substrate binding to rat liver fatty acid synthetase.

Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.     

Effects of vitamin E and selenium deficiency on the fatty acid composition of rat retinal tissues.

The fatty acid composition of retinal tissues was measured in rats maintained for 26--32 weeks on each of the following diets: a purified basal diet deficient in alpha-tocopherol and selenium, an identical control diet supplemented with alpha-tocopherol and selenium, and a commerical laboratory rat chow. Dietary deficiencies of antioxidant nutrients were found to cause a large decrease in total polyunsaturated fatty acids in the retinal pigment epithelium, a small decrease in the retinal rod outer segments, but no change in the whole retina or liver when compared to tissues from animals fed the vitamin E- and selenium-supplemented control diet. The polyunsaturated fatty acid content which we have observed for the retinal pigment epithelium from rats fed commerical lab chow is similar to that which we observed for bovine retinal pigment epithelium. Our results indicate that changes in fatty acid composition are not generalized to all tissues in severely antioxidant-deficient animals, but that changes do occur in some tissues, such as the retinal pigment epithelium, which appears to be particularly sensitive to in vivo lipid peroxidation.     

Changes in fatty acid composition of phospholipids and triacylglycerols after cold-acclimation of an aestivating insect prepupa

Prepupae of the Mediterranean arctiid moth Cymbalophora pudica spend hot spring and summer months in a summer diapause (aestivation). Although their cold-hardiness (survival after 1-day exposure to subzero temperatures) is relatively low, it may be moderately enhanced by prior cold acclimation at decreasing above-zero temperatures. In this study, fatty acids of phospholipids and triacylglycerols were analysed in five different tissues (body wall, midgut, fat body, silk glands and brain) dissected from both cold-acclimated and control aestivating prepupae. The five most abundant fatty acids (oleic, palmitic, stearic, linoleic and α-linolenic), found generally in both lipidic fractions and all five tissues, represent a typical fatty complement of Lepidoptera. The fatty acid profiles of individual tissues differed from each other and the response to cold acclimation was also tissue-specific. Moderate but significant increases in the proportion of unsaturated fatty acids after cold acclimation were observed in triacylglycerols of the body wall, fat body and silk glands. Additionally, significant rearrangements of fatty acid profiles were found in triacylglycerols of midgut and brain, without changing the unsaturation/saturation ratio. The adaptational value of enhanced fluidity of fat body triacylglycerols caused by their increased unsaturation remains unclear, because the lipidic energy depots are not utilized during aestivation of this insect. Minimal capacity to alter the membrane-bound fatty acids was found in all tissues except midgut, where the unsaturation/saturation ratio of phospholipids slightly increased after cold acclimation. A low ability to alter the composition of membrane lipids in response to low temperature, correlates well with the low capacity of C. pudica prepupae to enhance their cold-hardiness during cold-acclimation. This may be regarded as indirect support for the membrane lipid restructuring in insect cold adaptation. Accepted: 11 May 1998     

Stereospecific biosynthesis of triacylglycerols in mammary glands from lactating rats.

Microsomal plus cytosol preparations from the mammary gland of lactating rats are capable of incorporating palmitic acid and oleic acid into triacylglycerols. These triacylglycerols are similar in structure to those found in rat milk, where palmitic acid tends to be confined to the sn-2-position of the glycerol. Both glycerol 3-phosphate and dihydroxyacetone phosphate function as acyl acceptors. The enzymic synthesis of triacylglycerols appears in late pregnancy, increases rapidly during early lactation, but disappears within 3 days of weaning.     

The determination of the asymmetrical stereochemical distribution of fatty acids in triacylglycerols

The fatty acid distribution in triacylglycerols (TAGs) is a factor that contributes to the intrinsic properties of oils from different species variants. Many hypotheses have been proposed to explain the specific distribution of fatty acids in the different naturally occurring oils. Currently, the 1,3-random-2-random theory is more or less accepted, but it has been widely shown that most vegetable oils do not behave randomly in the sn-1 and sn-3 stereochemical positions. For this reason, complex methodologies have been developed to analyze the fatty acid composition of the three stereochemical positions in TAGs. In this article, we propose that by calculating the asymmetric alpha coefficient, the stereochemical asymmetry of fatty acids in TAG molecular species can be defined. This coefficient reflects the relative content of fatty acids at the sn-1 and sn-3 positions and may overcome the problems found mainly with complex sn-1, sn-2, and sn-3 stereochemical analysis of fatty acids in TAG. The alpha coefficient is calculated from the fatty acid, sn-2 fatty acid, and TAG composition of the oil. Indeed, through this coefficient, it has been possible to show that, despite having the same overall content, the stearic acid distribution in the sn-1 and sn-3 positions is not random in some oils.     

Effect of different levels of dietary fats upon the positional distribution of fatty acids in rat body triacylglycerols

• 1.1. The main purpose of the study was to describe and to compare the effect of different dietary fatty acids introduced at different levels into the diet on the positional distribution of fatty acids in rat body triacylglycerols. Only distribution between sn-2 (internal) and sn-1 + sn-3 (external) positions was considered in this study.
• 2.2. The positional distribution of fatty acids was first determined for controls fed on a low fat diet (1% D.M.). The same study was then carried out with 11 different dietary treatments and results were systematically compared with controls. The effects of four main molecules were tested: linoleic acid, oleic acid, lauric acid and capric acid.
• 3.3. Different alterations of the positional distribution of fatty acids in body triacylglycerols were obtained. They are graphically described and discussed.

     

Stereospecific analysis of triacylglycerols via racemic phosphatidylcholines and phospholipase C.

A method of simultaneous determination of stereospecific distribution and molecular association of acyl groups in triacylglycerols has been developed. The analysis is based on a random generation of rac-1,2-diacylglycerols by Grignard degradation, synthesis of rac-phosphatidylcholines, and a stereospecific stepwise release of 1,2-sn- and 2,3-sn-diacylglycerols by phospholipase C. The exact structure of the original triacylglycerols is reconstituted on the basis of complete analysis of the molecular species of the 1,2-sn- and 2,3-sn-diacylglycerols as the tertiary-butyldimethylsilyl ethers by gas chromatography with mass spectrometry. The validity of the method is demonstrated by analyses of synthetic triacylglycerols of known structure. A practical application is illustrated by determination of the fatty acid distribution in lard.