Turnover of free fatty acids during recovery from exercise.

The turnover of plasma free fatty acid (FFA) was studied during the recovery from exercise with the aid of a continuous infusion of 14C-labeled oleic acid. Arterial FFA reached a maximum of twice the exercise value after 6 min of recovery and was still 75% above the basal level after 20 min. Within 2 min after exercise, plasma radioactivity had increased and the specific activity of plasma oleic acid had fallen. The rate of uptake of FFA from the plasma pool rsoe by 40% during the first minutes after exercise. The rate of release of FFA to the plasma pool showed a peak 2 min after exercise and was thereafter about 40 mumol/min lower than the rate of uptake. The fractional turnover of FFA decreased to resting levels within 5-10 min after exercise. It is concluded that the postexercise peak in arterial FFA is a consequence of augmented release of FFA into the plasma pool above the level during exercise, possibly related to the release of sympathetic vasoconstrictor tone. As a consequence, the rate of removal of FFA rises at the end of exercise and remains augmented above the basal level for as long as the arterial concentration is increased.     

Cardiac triacylglycerol content and lipase activity during recovery from exercise

Female rats swam for 2-h to determine the temporal relationship between triglyceride (TG) repletion and TG lipase activity in the heart during recovery from exercise. Immediately after the exercise, plasma free fatty acids (FFA) had increased from a resting value of 0.44 +/- 0.04 to 0.84 +/- 0.04 mM. Heart TG concentration was reduced 75%, whereas the glycogen level was decreased 34% below control. TG lipase activity was elevated 33% above control activity. One hour after the end of the exercise, lipolytic activity was still 26% above control and did not return to the resting level until the 4th h of recovery. The cardiac TG concentration was back to control levels by the 2nd h after the swim. Plasma FFA concentrations remained elevated during the first 4 h of recovery and were back to the control level by h 8. Cardiac glycogen was "supercompensated" during recovery h 1 and 2 and returned to the preexercise level by h 4. These data indicate that TG is being synthesized in the heart while lipolytic enzyme activity is elevated above control levels. This points out that the rate of TG synthesis is in excess of the hydrolysis. Since plasma FFA concentrations are elevated during periods of augmented TG synthesis, substrate availability, namely plasma FFA, may play a key role in regulating the size of the intracellular TG pool.     

Transfer of fluorescent fatty acids from liver and heart fatty acid-binding proteins to model membranes

Fatty acid binding proteins (FABP) are a family of 14-15 kDa proteins found in high abundance in many mammalian cell types. The physiological functions of the FABP remain unknown. It is also not known whether each FABP has a unique function, or whether all FABP function in a similar manner in their respective tissues. In this report the rate of transfer of anthroyloxy-labeled free fatty acid (ffa) from FABP to phospholipid bilayers is monitored using a fluorescence resonance energy transfer assay. A comparison is made between heart muscle FABP and liver FABP, and the results show that the rate of ffa transfer from the heart protein is an order of magnitude greater than the rate of transfer from the liver protein. Ffa transfer rates from both liver and heart FABP are independent of acceptor concentration and composition, suggesting that, at least in the case of model membrane acceptor vesicles, the mechanism of transfer is via aqueous diffusion rather than via collision of FABP with membranes. Since the rate of ffa transfer is likely to be important to cellular ffa traffic, these studies suggest that heart FABP may function differently within the myocyte than does liver FABP within the hepatocyte.     

Response of algae and zooplankton to C18 fatty acids of Chlamydomonas reinhardtii

Three C₁₈ fatty acids were assayed for their activity against a number of algae and zooplankton. The three acids, lenolenic, lenoleic, and oleic, reduced the growth of Haematococcus lacustris, Synechococcus leopoliensis, and Botrydiopsis alpina by 50% of control growth in concentrations below 7 ppm. Calanoid and cyclopoid copepods in mixed cultures inoculated with lenolenic and lenoleic acid had LD₅₀ values below 10 ppm. An increase in copepod mortality was observed with increases in cyclopoid density and decreased with increases in calanoid density. Eucyclops agilis inoculated with lenolenic acid had a LD₅₀ value of 4 ppm.     

Regulation of triacylglycerol and phospholipid trafficking by fatty acids in newborn swine enterocytes

We (Wang H, Berschneider HM, Du J, and Black DD. Am J Physiol Gastrointest Liver Physiol 272: G935-G942, 1997; Wang H, Lu S, Du J, Yao Y, Berschneider HM, and Black DD. Am J Physiol Gastrointest Liver Physiol 280: G1137-G1144, 2001) previously showed that different fatty acids influence synthesis and secretion of triacylglycerol (TG) and phospholipid (PL) in a newborn swine enterocyte cell line (IPEC-1). The most striking effects were produced by stearic acid (SA; 18:0), which modestly affected TG and PL synthesis but reduced TG and PL secretion, and by eicosapentaenoic acid (EPA; 20:5), which reduced TG and PL synthesis and TG secretion relative to oleic acid (OA; 18:1). To define the mechanism of these effects, differentiated IPEC-1 cells were incubated for 24 h with OA, SA, or EPA and [(3)H]glycerol. Endoplasmic reticulum (ER) and Golgi (G) content of labeled lipids and apolipoprotein (apo) B and apoAI protein were measured. Relative to OA, SA did not impair ER TG synthesis, but reduced movement of labeled TG from ER to G. EPA impaired both ER TG synthesis and movement of labeled TG from ER to G. PL followed the same pattern, except ER synthesis of PL was relatively unaffected by EPA. Carbonate treatment demonstrated decreased partitioning of labeled lipid from ER membrane to lumen in EPA-treated cells. Organelle apoB and apoAI content demonstrated opposite patterns after SA and EPA incubation. We conclude that SA and EPA adversely influence immature enterocyte ER to G lipid trafficking, compared with OA. Furthermore, EPA inhibits ER lipid synthesis and transfer of membrane lipid to luminal particles. Regulation of apoAI ER to G trafficking is independent of that of apoB.     

Changes in the triacylglycerol content of mitochondrial membranes during development

The lipid content of mitochondria and mitochondrial membranes isolated from foetal, suckling, and adult rat liver mitochondria were compared. In foetal liver mitochondria triacylglycerol made up 26% of the lipids, while in adult rat liver mitochondria the triacylglycerol content was 7%. Esterified fatty acids originating from non-phospholipid sources amounted to 34% in mitochondrial membranes, and this amount decreased in mitochondrial membranes of adult rat liver to 22%. The concentration of phospholipids in the mitochondrial membranes did not change significantly during development.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms

Intestinal enterocytes contain high concentrations of two cytosolic fatty acid-binding proteins (FABP), liver FABP (L-FABP) and intestinal FABP (I-FABP), which are hypothesized to play a role in cellular fatty acid trafficking. The mechanism(s) by which fatty acids move from membranes to each of these proteins is not known. Here we demonstrate that fluorescent anthroyloxy fatty acid analogues (AOFA) are transferred from phospholipid vesicles to L-FABP versus I-FABP by different mechanisms. For L-FABP a diffusion-mediated transfer process is demonstrated. The AOFA transfer rate from phosphatidylcholine-containing vesicles (POPC) to L-FABP is similar to that observed with another diffusional process, namely inter-membrane AOFA transfer. Furthermore, the AOFA transfer rate was modulated by buffer ionic strength and AOFA solubility, while the transfer rate remained relatively unchanged by the presence of anionic phospholipids in vesicles. In contrast, the data for I-FABP suggest that a transient collisional interaction of I-FABP with the phospholipid membrane occurs during AOFA extraction from the vesicles by the protein. In particular, the presence of the anionic phospholipid cardiolipin in donor vesicles increased the rate of AOFA transfer to I-FABP by 15-fold compared with transfer to POPC vesicles. The effects of ionic strength on transfer suggest that the interaction of I-FABP with cardiolipin-containing vesicles is likely to contain an electrostatic component. Finally, based on the regulation of AOFA transfer to I-FABP compared with transfer from I-FABP, it is hypothesized that apo- and holo-I-FABPs adopt conformations which may differentially promote I-FABP-membrane interactions.In summary, the results suggest that I-FABP, but not L-FABP, can directly extract fatty acids from membranes, supporting the concept that I-FABP may increase the cytosolic flux of fatty acids via intermembrane transfer.     

Continuous changes in triacylglycerol molecular species composition by fatty acids in porcine adipocytes

It has been demonstrated that the composition of molecular species of adipose tissue triacylglycerols (TGs) from farm animals are not equally synthesized and that some molecular species are preferentially synthesized. The objective of the present study was to determine whether exogenous fatty acids (FAs) would affect the TG composition. To this end, the composition of TG molecular species stored in porcine adipocytes differentiated with several long-chain FAs was analyzed by gas chromatography. The addition of each FA for 6 d increased TG molecular species having two or three added FAs. However, the molecular species compositions at 15 d after the addition of each FA resembled those of cells with no added FAs. Moreover, some common molecular species in all experimental cells increased, as well as cells with no added FAs. It was concluded that the addition of FAs increases the contents of specific molecular species, but does not affect the synthetic processes of individual TG molecular species.     

Regulation of triacylglycerol hydrolase expression by dietary fatty acids and peroxisomal proliferator-activated receptors

Triacylglycerol hydrolase (TGH) is an enzyme that catalyzes the lipolysis of intracellular stored triacylglycerol (TG). Peroxisomal proliferator-activated receptors (PPAR) regulate a multitude of genes involved in lipid homeostasis. Polyunsaturated fatty acids (PUFA) are PPAR ligands and fatty acids are produced via TGH activity, so we studied whether dietary fats and PPAR agonists could regulate TGH expression. In 3T3-L1 adipocytes, TGH expression was increased 10-fold upon differentiation, compared to pre-adipocytes. 3T3-L1 cells incubated with a PPARγ agonist during the differentiation process resulted in a 5-fold increase in TGH expression compared to control cells. Evidence for direct regulation of TGH expression by PPARγ could not be demonstrated as TGH expression was not affected by a 24-h incubation of mature 3T3-L1 adipocytes with the PPARγ agonist. Feeding mice diets enriched in fatty acids for 3 weeks did not affect hepatic TGH expression, though a 3-week diet enriched in fatty acids and cholesterol increased hepatic TGH expression 2-fold. Two weeks of clofibrate feeding did not significantly affect hepatic TGH expression or microsomal lipolytic activities in wild-type or PPARα-null mice, indicating that PPARα does not regulate hepatic TGH expression. Therefore, TGH expression does not appear to be directly regulated by PPARs or fatty acids in the liver or adipocytes.     

Structured triacylglycerol containing medium-chain fatty acids in sn-1(3) facilitates the absorption of dietary long-chain fatty acids in rats

A study was carried out to examine if the positional distribution of medium chain fatty acids (MCF) in triacylglycerol influences dietary fat absorption in rats. Two types of structure-specific fats, one predominantly composed of MCF in sn-1(3) and iinoleic acid in sn-2 [sn1(3)MCF-structured] and the others of MCF in sn-2 and linoleic acid in sn-1(3) [sn-2MCF-structured], were initially prepared, and the two structure-specific fats were interesterified and designated as sn-1(3)MCF-interesterified and sn-2MCF-interesterified. Synthetic fat was mixed with an equal amount of cocoa butter (103 g/kg of diet) and was supplemented to the AIN93G-based diet. Rats were fed on the diets for 4 wk. Long-chain saturated fatty acids were the predominant fatty acids excreted into the feces, and the positional distribution of MCF resulted in an altered fat absorption rate (%) of 81.8, 82.5, 84.2 and 86.3 for the rats fed on the diets containing sn-2MCF-structured, sn-1(3)MCF-interesterified, sn-2MCF-interesterified and sn-1(3)MCF-structured fats, respectively. The proportion of MCF in the serum, liver and adipose tissue triacylglycerols was not affected by the MCF distribution of the dietary fats. These results indicate that the distribution of MCF in dietary triacylglycerol is a determinant of intestinal fat absorption.     

Synthesis of cyclopropane fatty acids in isolated bacterial membranes

Isolated E. coli membrane vesicles can synthesize cyclopropane fatty acids from S-adenosylmethionine using endogenous membrane phospholipid as the lipidsubstrate. The major methylated products are methylenehexadecanoic acid and methyleneoctadecenoic acid esterified to phosphatidylethanolamine. The membrane system is slightly stimulated by sodium dodecyl sulfate at low concentrations but is inhibited by neutral surfactants. The reaction is inhibited by phospholipase C and phospholipase A. The Arrhenius plot for the enzyme reaction is discontinuous over the temperature range 0–35 °C.     

Changes in triacylglycerol, diacylglycerol and free fatty acids after fertilization in developing toad embryos

The content and fatty-acid composition of triacylglycerols, diacylglycerols and free fatty acids were analyzed from the unfertilized oocyte stage to the gastrula stage in the toad Bufo arenarum Hensel. Fertilization triggered a 30% and a 40% decrease in triacylglycerol and diacylglycerol, respectively. In contrast, free fatty acid increased continuously from oocyte to gastrula stage with an accumulation of palmitate predominating. However, the observed increase in free fatty acid was too small to account for the decreases in both neutral glycerides. The decrease in triacylglycerol might be a reflection of the activation of lipolytic enzymes and the subsequent oxidation of fatty acids to meet the increased metabolic energy requirements brought on by fertilization. The diminished diacylglycerol content due to fertilization may be accounted for, at least in part, by the utilization of diacylglycerol in the synthesis of membrane phospholipids, inasmuch as their decrease occurred simultaneously with an increase in phosphatidic acid. When cell-free homogenates taken from the three stages of development (unfertilized, fertilized and gastrula) were incubated in Tris-Ringer buffer for 90 min, free fatty acid accumulated. Triacylglycerol and diacylglycerol did not change substantially during this incubation period. This fact indicates that the free fatty acid released during incubation was not derived from neutral glycerides, but probably from membrane phospholipids. The release of free fatty acid was significantly greater in samples from the fertilized oocyte stage. The results described in this paper suggest that the synthesis of membrane phospholipids, including an enhanced turnover of the acyl moiety, plays a significant role in the metabolic events activated by fertilization.     

Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs

The effect of different extraction techniques on the recovery of fatty acids from freeze-dried biomass of two lipid-producing microheterotrophs was examined. Two procedures were used: the extraction of lipids from biomass followed by transesterification of the fatty acids (extraction-transesterification); and the direct transesterification of biomass to produce fatty acid methyl esters (i.e. without the initial extraction step). Variable factors in the extraction-transesterification experiment were the sequence in which solvents were added to the samples, the relative amount of methanol in the solvent mix, and sonication of biomass while in the solvent mix. Variable factors in the direct transesterification experiment were sample size, and reaction duration. Statistical analysis of data (level of significance P<0.05) showed that: (1) extraction of total fatty acids prior to transesterification was significantly more efficient when solvents were added in the order of increasing polarity; (2) neither sonication nor increasing the proportion of methanol in the extraction solvent significantly affected extraction of fatty acids prior to transesterification; (3) efficiency of direct transesterification of fatty acids increased significantly with reaction time; (4) efficiency of direct transesterification of fatty acids was not significantly affected by sample size; (5) the most efficient method for extraction of fatty acids prior to transesterification yielded significantly less fatty acids than the most effective direct transesterification method. While the study examined only two strains, our results suggest that fatty acid analysis methodology for microheterotrophs under consideration for biotechnological exploitation requires optimisation and validation.     

Randomly interesterified triacylglycerol containing medium- and long-chain fatty acids stimulates fatty acid metabolism in white adipose tissue of rats

We have reported previously that randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule (MLCT) resulted in significantly lower body fat accumulation and higher hepatic fatty acid oxidation than from long-chain triacylglycerol (LCT) in rats. To understand the metabolic changes occurring in white adipose tissue, the fatty acid oxidation and synthesis, and the adipocytokine level were measured in rats fed with MLCT or LCT for 2 weeks. In comparison with LCT, MLCT lowered not only the fatty acid synthase and glycerol-3-phosphate dehydrogenase activities in perirenal adipose tissue, but also the serum insulin and leptin levels, in addition to significantly reducing the body fat accumulation. In contrast, fatty acid oxidation measured as the carnitine palmitoyltransferase activity in the tissue was significantly higher in the MLCT-fed rats than in the LCT-fed rats. It seems that the altered fatty acid metabolism in adipose tissue per se was also responsible for the lower adiposity by dietary MLCT.