Saturated fatty acids induce c-Src clustering within membrane subdomains, leading to JNK activation

Saturated fatty acids (FA) exert adverse health effects and are more likely to cause insulin resistance and type 2 diabetes than unsaturated FA, some of which exert protective and beneficial effects. Saturated FA, but not unsaturated FA, activate Jun N-terminal kinase (JNK), which has been linked to obesity and insulin resistance in mice and humans. However, it is unknown how saturated and unsaturated FA are discriminated. We now demonstrate that saturated FA activate JNK and inhibit insulin signaling through c-Src activation. FA alter the membrane distribution of c-Src, causing it to partition into intracellular membrane subdomains, where it likely becomes activated. Conversely, unsaturated FA with known beneficial effects on glucose metabolism prevent c-Src membrane partitioning and activation, which are dependent on its myristoylation, and block JNK activation. Consumption of a diabetogenic high-fat diet causes the partitioning and activation of c-Src within detergent insoluble membrane subdomains of murine adipocytes.     

Different fatty acid composition in central and peripheral adipose tissues of the American mink (Mustela vison)

Fatty acid (FA) composition in the intraabdominal (IAB), subcutaneous (SC) and peripheral adipose tissues of the semiaquatic American mink (Mustela vison) was examined in comparison to the diet by gas-liquid chromatography. There was a clear compositional gradient from the IAB via SC to peripheral adipose tissues and the anatomically different adipose tissues accumulated or metabolized FA selectively. The total lipids of the body appendages had smaller proportions of saturated (SFA) and larger proportions of monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) than the lipids of the trunk adipose tissues. Especially n-3 PUFA were enriched in the periphery. The appendages were also characterized with a high ratio of unsaturated FA to SFA, an increased Delta9-desaturation index and increased mean numbers of double bonds and carbon atoms in a FA molecule. The proportions of SFA and MUFA of the diet resembled the trunk adipose tissues while the dietary percentage of n-3 PUFA surpassed those of the trunk fat depots but was lower than those of the peripheral fats. These data confirm that the FA signatures of mammals reflect not only their dietary history but also metabolic modifications of ingested FA.     

Formation of organic compounds from simulated Titan atmosphere: perspectives of the Cassini mission.

Gas mixtures of methane and nitrogen were subjected to proton irradiation (PI), gamma irradiation (GI), UV irradiation (UV) or spark discharges (SD), and the products were analyzed to compare possible energy sources for synthesis of organics in Titan. SD mainly gave unsaturated hydrocarbons, while PI gave saturated hydrocarbons. N-containing organics were detected in PI, GI and SD, but not in UV. The formers yielded amino acids after acid-hydrolysis of solid phase products (tholin). Comparison of the present results with those by Cassini-Huygens [correction of Heygens] mission will make it possible to prove major energy sources for organic synthesis in Titan atmosphere.     

DAG accumulation from saturated fatty acids desensitizes insulin stimulation of glucose uptake in muscle cells

The increased availability of saturated lipids has been correlated with development of insulin resistance, although the basis for this impairment is not defined. This work examined the interaction of saturated and unsaturated fatty acids (FA) with insulin stimulation of glucose uptake and its relation to the FA incorporation into different lipid pools in cultured human muscle. It is shown that basal or insulin-stimulated 2-deoxyglucose uptake was unaltered in cells preincubated with oleate, whereas basal glucose uptake was increased and insulin response was impaired in palmitate- and stearate-loaded cells. Analysis of the incorporation of FA into different lipid pools showed that palmitate, stearate, and oleate were similarly incorporated into phospholipids (PL) and did not modify the FA profile. In contrast, differences were observed in the total incorporation of FA into triacylglycerides (TAG): unsaturated FA were readily diverted toward TAG, whereas saturated FA could accumulate as diacylglycerol (DAG). Treatment with palmitate increased the activity of membrane-associated protein kinase C, whereas oleate had no effect. Mixture of palmitate with oleate diverted the saturated FA toward TAG and abolished its effect on glucose uptake. In conclusion, our data indicate that saturated FA-promoted changes in basal glucose uptake and insulin response were not correlated to a modification of the FA profile in PL or TAG accumulation. In contrast, these changes were related to saturated FA being accumulated as DAG and activating protein kinase C. Therefore, our results suggest that accumulation of DAG may be a molecular link between an increased availability of saturated FA and the induction of insulin resistance.     

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Fatty acid composition of total lipids from the needles of pine seedlings as related to boron availability

The effect of boron availability on the content of fatty acids (FA) in the total lipids of needles was investigated in two-year-old seedlings of Scots pine (Pinus sylvestris L.). The study was conducted in the forest nursery on the soil deficient in boron. Various rates of boric acid (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/m²) were applied to soil three times throughout the growth period. When pine seedlings were supplied with extra boron, the lipid content of the seedling needles declined. The optimal boron supply elevated the content of saturated FA in total needle lipids mostly at the expense of palmitic acid, with a corresponding decline in the level of unsaturated FA and unsaturation index of FA owing to trienoic FA (mainly linolenic and hexadecatrienoic acids). At the same time, the level of monoenoic and to a lesser degree dienoic acids increased. When boron supply of the seedlings was not optimal, the content of unsaturated and low-molecular FA increased.     

Protective role of mitochondrial unsaturated lipids on the preservation of the apoptotic ability of cytochrome C exposed to singlet oxygen

Cytochrome c-mediated apoptosis in cells submitted to photodynamic therapy raises the question about the ability of photodynamically oxidized cytochrome c (cytc405) to trigger apoptosis as well as the effect of membranes on protein photo-oxidation. Cytochrome c was submitted to irradiation in the presence of MB+ in phosphate buffer and in the presence of four types of phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PCPECL) liposomes (50/30/20%): totally saturated lipids (tsPCPECL), totally unsaturated lipids (tuPCPECL), partially unsaturated (80%) lipids, with unsaturation in the PC and PE content (puPCPECL80), and partially unsaturated (20%) lipids, with unsaturation in the CL content (puPCPECL20). Cytc405 was formed by irradiation in buffered water and in tsPCPECL and puPCPECL20 liposomes. In the presence of tuPCPECL and puPCPECL80, cytochrome c was protected from photodynamic damage (lipid-protected cytochrome c). In CL liposomes, 25% unsaturated lipids were enough to protect cytochrome c. The presence of unsaturated lipids, in amounts varying according to the liposome composition, are crucial to protect cytochrome c. Interesting findings corroborating the unsaturated lipids as cytochrome c protectors were obtained from the analysis of the lipid-oxidized derivatives of the samples. Native cytochrome c, lipid-protected cytochrome c, and cytc405 were microinjected in aortic smooth muscle cells. Apoptosis, characterized by nucleus blebbing and chromatin condensation, was detected in cells loaded with native and lipid protected cytochrome c but not in cells loaded with cytc405. These results suggest that photodynamic therapy-promoted apoptosis is feasible due to the protective effect of the mitochondrial lipids on the cytochrome c structure and function.     

Effect of alternating magnetic field on the composition and level of lipids in radish seedlings

The effects of alternating magnetic field (AMF) with the frequency of 50 Hz on the dynamics of unfolding of cotyledon leaves, the composition and level of polar and neutral lipids and their component fatty acids (FA) were studied in 5-day-old radish seedlings (Raphanus sativus L. var. radicula D.L., cv. Rozovo-krasnyi s belym konchikom) grown in the light and in the dark. AMF weakened the inhibitory effect of light on unfolding of cotyledon leaves. In the light, the total content of lipids, as well as the level of polar and neutral lipids, in the seedlings in AMF was greater than in control material. In polar lipids, the total amount of glyco-and phospholipids increased; in neutral lipids, the level of triacylglycerols rose. The ratio between phospholipids and sterols (PhL/S) increased. In the dark, the total content of lipids and the level of neutral lipids in the seedlings in AMF were lower than in control material, and the ratio PhL/S decreased. In control material, there were no differences in the relative total content of unsaturated FA in the light and in the dark, whereas the level of linolenic acid was higher in the light than in the dark. AMF induced a decrease in the content of linolenic acid in the light and a rise in the dark; the level of erucic acid in the light decreased. The ratio between unsaturated and saturated FA decreased both in the light and in the dark. It was concluded that AMF with the frequency of 50 Hz was an adjusting agent considerably changing the content of lipids in the radish seedlings in the light and in the dark.     

Cellular responses to unsaturated fatty acids mediated by their sensor Ubxd8

Fatty acids (FAs) are crucial nutrient for cell survival because they are required for synthesis of phospholipids in cellular membranes and for generation of energy. However, overaccumulation of FAs is toxic. In response to excessive FAs, cells are able to activate multiple reactions to prevent their overaccumulation. These reactions are mediated by Ubxd8, a cellular protein that specifically interacts with unsaturated but not saturated FAs. The selective interaction of Ubxd8 with unsaturated FAs may explain previous observations that only unsaturated but not saturated FAs are able to stimulate the regulatory reactions that prevent overaccumulation of FAs. Thus, understanding the mechanism through which Ubxd8 maintains cellular FA homeostasis may provide new insights into saturated FAinduced lipotoxicity.     

Changes in fatty acid composition of lipids in chloroplast membranes of tobacco plants during cold hardening

Changes in fatty acid composition of chloroplast membrane lipids were investigated using tobacco (Nicotiana tabacum L., cv. Samsun) plants subjected to cold hardening for 6 days at 8°C. Under optimal growing temperature (22°C), the lipids of thylakoid membranes were characterized by elevated content of 16:3n-3 and 18:3n-3 fatty acids (FA). Compared to the lipids of chloroplast envelope membranes, the thylakoid lipids were less rich in the content of saturated, mono- and diunsaturated FA. The relative content of unsaturated FA in chloroplast membranes increased substantially during cold hardening, which was mainly due to the accumulation of 18:3n-3 FA. It is concluded that the observed changes in FA composition of chloroplast lipids during cold hardening adjust the fluidity of these membranes to the level sufficient for functioning of tobacco photosynthetic apparatus, which is a prerequisite for accumulation of assimilates and allows the hardened tobacco plants to survive under conditions of hypothermia.     

Changes in the content and composition of lipid fatty acids in tobacco leaves and roots at low-temperature hardening

Changes in the fatty acid (FA) composition of leaf and root lipids of heat-loving tobacco (Nicotiana tabacum L., cv. Samsun) plants during low-temperature hardening (8°C for 6 days) were studied. Hardening could improve leaf but not root cold tolerance. As this took place, the relative content of polyunsaturated (18:2n-6 and 18:3n-3) FAs increased and the proportion of saturated and monounsaturated FAs decreased. In contrast, in the roots hardening slightly increased the concentration of saturated FAs (16:0 and 18:0) and reduced the level of unsaturated FAs (18:1n-9, 18:2n-6, and 18:3n-3). At the same time, root lipids contained much C_20–24 FAs, and their content increased during hardening. It was suggested that an increased FA saturation and elevated proportion of C_20–24 FAs in the root lipids resulting in the lower membrane fluidity could be a reason for incapability of heat-loving tobacco plant roots of hardening and plant death at the lowtemperature stress.     

Cuticular lipids and odors induce sex-specific behaviors in the male cricket Gryllus bimaculatus

Male crickets display sex-specific (e.g., mating and agonistic) behaviors towards conspecific individuals. One of the key signals for these behaviors is the chemical substance on the cricket body surface. In the present study, we analyzed female and male cuticular substances in behavioral assays. Antennal contact stimulation using female forewings elicited a mating behavior in males, while that using male forewings elicited an agonistic behavior in males. Thin-layer-chromatographic and other techniques analysis showed that saturated cuticular lipids were present in both female and male cuticles and that unsaturated lipids were present only in the male cuticle. Filter papers soaked with saturated or unsaturated cuticular lipids were applied to antennae of male crickets. Males showed mating behavior in response to stimulation with saturated lipids from both females and males but showed avoidance behavior in response to stimulation with male unsaturated lipids. Because cuticular lipids did not induce agonistic behavior in males, we collected odors from male crickets and found that these odors induced agonistic behavior in males. Therefore, we concluded that the key signals for mating, avoidance and agonistic behaviors of male crickets are comprised of at least three different components, saturated and unsaturated cuticular lipids and male odors, respectively.     

Antibacterial actions of fatty acids and monoglycerides against Helicobacter pylori

The bactericidal potencies of saturated and unsaturated fatty acids (FAs) and monoglycerides (MGs) against Helicobacter pylori were determined following short incubations with freshly harvested cells over a range of pHs. FAs and their derivatives with an equivalent-carbon number of 12 were the most potent: lauric acid had a minimum bactericidal concentration (MBC) at pH 7.4 of 1 mM, myristoleic and linolenic acid were the most potent unsaturated FAs (MBCs of 0.5 mM, pH 7.4), and monolaurin was the most potent MG (MBC 0.5 mM). Potencies of saturated FAs were increased sharply by lowering pH, and a decrease of only 0.5 pH units can cause a change from non-lethal to lethal conditions. Conversely, the bactericidal action of monolaurin was not pH-dependent. The bactericidal potencies of unsaturated FAs increased with degree of unsaturation. When more than one FA or FA plus MGs were present, their combined action was additive. Urea and endogenous urease did not protect H. pylori from the bactericidal action of FAs. These results suggest that H. pylori present in the stomach contents (but not necessarily within the mucus barrier) should be rapidly killed by the millimolar concentrations of FAs and MGs that are produced by pre-intestinal lipase(s) acting on suitable triglycerides such as milk fat.     

Free fatty acids associated with the high molecular weight aminoacyl-tRNA synthetase complex influence its structure and function

Aminoacyl-tRNA synthetases from higher eukaryotes often are isolated as high molecular weight complexes associated with other components such as lipids. Since hydrophobic interactions are involved in the organization of the complex, it has been suggested that interaction of synthetases with these lipids might be important for their structure and function. Delipidation is known to affect certain properties of synthetases within the complex including sensitivity to detergents plus salts, temperature inactivation, hydrophobicity, sensitivity to proteases, and, as shown here, sensitivity to p-mercuribenzoate and sites of papain cleavage. Of the lipids known to co-purify with the complex, cholesterol esters, phospholipids and free fatty acids, we show that the particular lipids responsible for many of these changes are the free fatty acids. Specific removal of fatty acids results in a complex with properties similar to one totally delipidated by detergent treatment, and readdition of the fatty acid fraction reverses the effects. The fatty acid fraction contains both saturated and unsaturated fatty acids, but unsaturated fatty acids are much more effective in reversing the properties of the delipidated complex that are saturated fatty acids. These results indicate that the free fatty acids co-purifying with the synthetase complex bind to the synthetases and affect their structure and function.     

Perturbation of DPPC/POPG bilayers by the N-terminal helix of lung surfactant protein SP-B: a <Superscript>2</Superscript>H NMR study

SP-B_8–25 is a synthetic peptide comprising the N-terminal helix of the essential lung surfactant protein SP-B. Rat lung oxygenation studies have shown that SP-B_8–25 retains some of the function of full-length SP-B. We have used deuterium nuclear magnetic resonance (²H-NMR) to examine the influence of SP-B_8–25 on the mixing properties of saturated PC and unsaturated PG lipids in model mixed lipid bilayers containing dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), in a molar ratio of 7:3. In the absence of the peptide, ²H-NMR spectra of DPPC/POPG mixtures, with one or the other lipid component deuterated, indicate coexistence of large liquid crystal and gel domains over a range of about 10°C through the liquid crystal to gel transition of the bilayer. Addition of SP-B_8–25 has little effect on the width of the transition but the spectra through the transition range cannot be resolved into distinct liquid crystal and gel spectral components suggesting that the peptide interferes with the tendency of the DPPC and POPG lipid components in this mixture to phase separate near the bilayer transition temperature. Quadrupole echo decay observations suggest that the peptide may also reduce differences in the correlation times for local reorientation of the two lipids. These observations suggest that SP-B_8–25 promotes a more thorough mixing of saturated PC and unsaturated PG components and may be relevant to understanding the behaviour of lung surfactant material under conditions of lateral compression which might be expected to enhance the propensity for saturated and unsaturated surfactant lipid components to segregate.     

The effect of dietary ethanol on the composition of lipids of Drosophila melanogaster larvae

At a moderate concentration (2.5%, v/v) dietary ethanol reduced the chain length of total fatty acids (FA) and increased the desaturation of short-chain FA in Drosophila melanogaster larvae with a functional alcohol dehydrogenase (ADH). The changes in length in total FA were postulated to be due to the modulation of the termination specificity of fatty acid synthetase. Because the ethanol-stimulated reduction in the length of unsaturated FA was blocked by linoleic acid, it was thought to reflect the properties of FA 9-desaturase. Although the ethanol-stimulated reduction in chain length of unsaturated FA was also observed in ADH-null larvae, ethanol promoted an increase in the length of total FA of the mutant larvae. Thus, the ethanolstimulated change in FA length was ADH dependent but the ethanol effect on FA desaturation was not. Ethanol also stimulated a decrease in the relative amount of phosphatidylcholine and an increase in phosphatidylethanolamine. Because similar ethanol-induced changes have been found in membrane lipids of other animals, ethanol may alter the properties of membranes in larvae. It is proposed that ethanol tolerance in D. melanogaster may be dependent on genes that specify lipids that are resistant to the detrimental effects of ethanol.This research was supported by National Institutes of Health Grant GM-28779 to B.W.G. and a Monash University Research Grant to S.W.M.     

Changes in fatty acid compositions of mitochondria during embryonic development

1. A general trend among biomembranes of hepatocytes in the developing avian embryo is to display increasing percentages of unsaturated fatty acids, especially oleic acid (C18:1). 2. However, once increasing amounts of thyroxine appear in the plasma, mitochondria begin to exhibit increasing percentages of saturated fatty acids, primarily stearic acid (C18:0). 3. Increasing saturation of mitochondrial membrane lipids can be inhibited by exposure of embryonated eggs to 500 R of X-irradiation. 4. Injection of embryonated eggs with estrone increases the proportion of oleic acid (C18:1) in mitochondrial membranes but a balancing increase in palmitic acid (C16:0) enables their lipids to remain more saturated than unsaturated.     

Fatty acid mobilization in voles--model species for rapid fasting response and fatty liver

Factors regulating fatty acid (FA) composition of small herbivores are poorly known. Because of the fast response to food deprivation, the tissue FA profiles of voles could be rapidly modified. The selectivity of incorporating dietary FA into tissue total lipids and mobilizing tissue FA was examined in two Microtus vole species either fed or fasted for 12-18 h. The FA composition of the tissues reflected the dietary lipids, but FA were selectively incorporated depending on their structure. The FA profiles of white and brown adipose tissues were different and contained more saturated and monounsaturated FA and less polyunsaturated FA (PUFA) than the diet. The essential PUFA precursors with smaller tissue percentages were likely converted into longer-chain derivatives for structural lipids. The FA composition of the vole tissues was selectively modified by food deprivation. The preferences for retention or loss were tissue-specific and related to the FA structure. Livers displayed steatosis with characteristic accumulation of triacylglycerols, while FA prevalent in membrane phospholipids decreased in proportion. Hepatic FA could be partly derived from lipids hydrolyzed in fat depots. The FA profiles of the vole tissues reflect the dietary lipids and are rapidly and selectively modified by food deprivation.