Free fatty acid inhibition of the insulin induction of glucose-6-phosphate dehydrogenase in rat hepatocyte monolayers

Rat hepatocytes in monolayer culture were utilized to determine if the decrease in glucose-6-phosphate dehydrogenase (G6PD) activity resulting from the ingestion of fat can be mimicked by the addition of fatty acids to a chemically, hormonally defined medium. G6PD activity in cultured hepatocytes was induced several-fold by insulin. Dexamethasone or T3 did not amplify the insulin induction of G6PD. Glucose alone increased G6PD activity in cultured hepatocytes from fasted donors by nearly 500%. Insulin in combination with glucose induced G6PD an additional two-fold. The increase in G6PD activity caused by glucose was greater in hepatocytes isolated from 72 hr-fasted rats as compared to fed donor rats. Such a response was reminiscent of the “overshoot” phenomenon in which G6PD activity is induced well above the normal level by fasting-refeeding rats a high glucose diet. Addition of linoleate to the medium resulted in a significant suppression of insulin’s ability to induce G6PD, but linoleate had no effect on the induction of G6PD activity by glucose alone. A shift to the right in the insulin-response curve for the induction of G6PD also was detected for the induction of malic enzyme and acetyl-CoA carboxylase. Arachidonate (0.25 mM) was a significantly more effective inhibitor of the insulin action than linoleate was. Apparently rat hepatocytes in monolayer culture can be utilized as a model to investigate the molecular mechanism by which fatty acids inhibit the production of lipogenic enzymes. In part, this mechanism of fatty acid inhibition involves desensitization of hepatocytes to the lipogenic action of insulin.     

Composition of fatty acids and structure of triglycerides in medium and low erucic acid rapeseed

Total triglycerides in medium (MEAR) and low (LEAR) erucic acid cultivars of rapeseed were fractionated by argentation chromatography into twelve and ten fractions, respectively. Gas liquid chromatography of the fatty acids in the triglyceride fractions and their 2-monoglycerides was used to evaluate the structural characteristics of the individual fractions. Fractionation occurred on the basis of degree of unsaturation, molecular weight and positional characteristics. The most mobile fractions contained 34–50% of saturated fatty acids while the less mobile had 59–65% of polyunsaturated fatty acids. In the medium erucic acid oil, long chain fatty acids (C20–C22) were found in all fractions, but four fractions of low erucic acid oil were essentially free of long chain acids. Two of these fractions in the latter oil, which represented 44% of the total triglycerides, were glycerol trioleate and dioleoyllinoleoylglycerol. The majority of the 2-positions were occupied by unsaturated C18 fatty acids, generally in the order of linoleic ≥linolenic> oleic acids. The saturated and long chain fatty acids occurred predominantly in the 1-and 3-positions. The various fractions of medium and low erucic acid oils were similar in structural composition except that eicosenoic and erucic acids substituted for oleic acid in some external positions. Erucic acid did not appear to substitute directly for oleic acid in the 2-position.     

Isomeric monoethylenic fatty acids in herring oil

Monoethylenic fatty acids from herring oil were concentrated by chromatography by chromatography on silver nitratesilicic acid columns. Examination of consecutive fractions by open tubular gas chromatography confirmed the preferential elution of longer chain length esters and of esters within one chain length with the double bond closer to the terminal methyl group. Isomeric monoethylenic fatty acids with double bonds in the positions closer to the carboxyl group than the approximate midpoint of the even-numbered fatty acid chains could not be adequately separated by gas chromatography and were determined by ozonolysis. The isomers observed are consistent with primary formation from saturated acids through the action of an enzyme specifically removing hydrogen atoms in positions Δ⁹ and Δ¹⁰ relative to the carboxyl group. Chain extension of particular monoethylenic isomers by two carbon atoms in the C₂₀ and longer chain lengths is apparently influenced by the position of the double bond. This work was carried out in partial fulfillment of MSc requirements at Dalhousie University.     

Analyse hoch ungesättigter Fettsäuremethylester

Analysis of Highly Unsaturated Methyl Esters of Fatty Acids The conditions for an optimum separation and identification of fatty acids having 0-6 double bonds were investigated. Thin-layer chromatographic separation of methyl esters of fatty acids on silver nitrate containing plates by a special technique in two different solvent systems enables a fractionation according to the degree of saturation as well as a resolution of highly unsaturated ω3 fatty acids from their ω6 positional isomers. Subsequent gas chromatography of individual fractions permits the examination of homologues with the same degree of saturation but different chain length.     

Pristanic acid is activator of peroxisome proliferator activated receptor alpha

lsoprenoid phytanic acid (3,7,11,15‐tetramethylhexadecanoic acid) is degraded in peroxisomes by α‐oxidation to pristanic acid (2,6,10,14‐tetramethylpentadecanoic acid) and then via β‐oxidation. Branched‐chain phytanic acid is an activator of the peroxisome proliferator activated receptor α (PPAR ) which in liver cells regulates expression of genes encoding peroxisomal and mitochondrial β‐oxidative enzymes as well as cytosolic/nuclear liver‐type fatty acid binding protein (L‐FABP). In this report we address the question whether pristanic acid also acts as activator of PPARα and thus mediates the expression of its catabolizing enzymes. In a first in vivo approach we fed pristanic acid for 14 days to wildtype mice and to mice lacking sterol carrier protein 2/sterol carrier protein x which Ieads to a phenotype having high concentrations of branched‐chain fatty acids. In either genotype, feeding pristanic acid was associated with a strong induction of peroxisomal β‐oxidation enzymes tested (acyl‐CoA oxidase, bifunctional enzyme, thiolase) as well as of L‐FABP. The link between pristanic acid and protein expression observed was established by carrying out assays for transactivation of PPARα in transfected HepG2 cells. In comparison to hypolipidemic drugs and to straight‐chain fatty acids known to be PPARα agonists, branched‐chain phytanic and pristanic acids were substantially stronger activators, pristanic acid being even superior to phytanic acid.     

Branched‐chain fatty acids as activators of peroxisome proliferator‐activated receptors

We observed earlier that phytanic acid activated subtype α of the peroxisome proliferator‐activated receptor (PPAR) via the cytosolic liver‐type fatty acid‐binding protein (L‐FABP). In a further search for minor lipid nutrients that interact with genes, we explored here the potential of branched‐chain fatty acids to serve as agonists for the PPAR subtypes α, β and γ in rodent and human molecular test systems. Beyond chlorophyll‐derived pristanic and phytanic acids, bacteria‐derived iso‐ and anteiso‐fatty acids and avian‐derived ‘uropygial’ fatty acids were tested by transactivation assay. In addition, we studied binding of these fatty acids to recombinantly expressed PPAR ligand binding domains (LBDs) and to L‐FABP by competition with fluorescent parinaric acid. In contrast to single methyl‐branched agonists, multi methyl‐branched fatty acids had high transactivation potentials in either test system; in addition, some agonists of the latter were highly subtype selective. Multi methyl‐branched chain fatty acids were superior activators of human PPARγ, a preference not seen in the murine test system. High‐affinity binding of isoprenoid‐derived pristanic and phytanic acids to PPARγ‐LBD and to L‐FABP was observed, and also of pristanic acid to PPARα‐LBD. Polyketidic uropygial fatty acids bound to PPARγ‐LBD only, though weakly. As both isoprenoid and polyketidic fatty acids showed high activation potentials, it became clear that binding data determined in vitro cannot predict biological activity as determined by transactivation assay. For pristanic acid, however, a signalling path similar to that found for phytanic acid can be concluded. Taken together, multi methyl‐branched fatty acids of the human food chain can affect cellular metabolism through regulating gene expression.     

Main constituents of rapeseed lecithin

Commercial rapeseed lecithin has been analyzed after separation by silicic acid column chromatography. Besides neutral oil (40%), four major constituents have been found, viz., phosphatidyl ethanolamine (18%), phosphatidyl inositol (8%), phosphatidyl choline (16%) and sterol glycosides (8%). Among the minor fractions lysophosphatidyl ethanolamine accounts for about 2%. The phosphatides are characterized by low erucic acid content and the major fatty acids are palmitic, oleic and linoleic acids.     

Fractional vacuum distillation of herring oil methyl esters

Methyl esters of a Canadian Atlantic herring oil containing 62% monoethylenic fatty acids were subjected to batch fractional distillation under vacuum on a pilot plant scale, to study the feasibility of fractionating fatty acid esters of marine oils of low iodine value into monounsaturated fractions with increased commercial value for industrial chemical uses. A total of 64 methyl ester fractions were collected and analyzed by gas liquid chromatography. Recoveries of the major saturated and monounsaturated acids were close to 100%, and some fractions contained over 90% of the desired 22:1 long chain monounsaturated acids. The short chain polyunsaturated acids were recovered in good yields, but the long chain highly unsaturated acids were recovered in yields of 60% or less due to oxidative and thermal decomposition in the particular apparatus employed. If small amounts of unsaturated acids are acceptable, fractional distillation of low iodine value marine oils could inexpensively supply fractions with high concentrations of methyl esters of longer chain (C₂₀ and C₂₂) monounsaturated and shorter chain (C₁₄) saturated acid or (C₁₆) saturated-monounsaturated acid mixture.     

Liver Fatty Acid Binding Protein Gene-Ablation Exacerbates Weight Gain in High-Fat Fed Female Mice

Loss of liver fatty acid binding protein (L‐FABP) decreases long chain fatty acid uptake and oxidation in primary hepatocytes and in vivo. On this basis, L‐FABP gene ablation would potentiate high‐fat diet‐induced weight gain and weight gain/energy intake. While this was indeed the case when L‐FABP null (?/?) mice on the C57BL/6NCr background were pair‐fed a high‐fat diet, whether this would also be observed under high‐fat diet fed ad libitum was not known. Therefore, this possibility was examined in female L‐FABP (?/?) mice on the same background. L‐FABP (?/?) mice consumed equal amounts of defined high‐fat or isocaloric control diets fed ad libitum. However, on the ad libitum‐fed high‐fat diet the L‐FABP (?/?) mice exhibited: (1) decreased hepatic long chain fatty acid (LCFA) β‐oxidation as indicated by lower serum β‐hydroxybutyrate level; (2) decreased hepatic protein levels of key enzymes mitochondrial (rate limiting carnitine palmitoyl acyltransferase A1, CPT1A; HMG‐CoA synthase) and peroxisomal (acyl CoA oxidase 1, ACOX1) LCFA β‐oxidation; (3) increased fat tissue mass (FTM) and FTM/energy intake to the greatest extent; and (4) exacerbated body weight gain, weight gain/energy intake, liver weight, and liver weight/body weight to the greatest extent. Taken together, these findings showed that L‐FABP gene‐ablation exacerbated diet‐induced weight gain and fat tissue mass gain in mice fed high‐fat diet ad libitum—consistent with the known biochemistry and cell biology of L‐FABP.     

Hydrierung von Lipiden als Hilfsmittel zur Identifizierung und Quantifizierung von Phosphatiden aus Membransystemen des Herzmuskels

Hydrogenation of Lipids for Identification and Quantification of Phosphatides from Pellicle Systems of Cardiac Muscle. It was the aim of our research to show that hydrogenation of lipids is an auxiliary technique in phospholipid analysis of cardiac membranes. This is of interest if a preliminary overview on lipid fractions containing highly unsaturated fatty acids is needed. The fatty acids and the diglycerides from phospholipids were hydrogenated according to the procedure described by Appelqvist (A simple and convenient procedure for the hydrogenation of lipids on the micro- and nanomole scale, J. Lipid Res. 13 (1972), 146) with platinum oxide as a catalyst. The lipids (fatty acid methyl esters or acetylated diglycerides) were taken to dryness in a test-tube under nitrogen and flushed with hydrogen. The catalyst, suspended in methanol was injected through a septum. For identification purposes thin-layer chromatography on silica gel and on silica gel impregnated with silver nitrate was combined with gas chromatography before and after hydrogenation. After hydrogenation the fatty acid profile is much simpler and the fatty acid methyl esters can easily be differentiated from dimethyl acetals, as the latter are more volatile. Diacylglycerides and alkenylacylglycerides were also separated by thin-layer chromatography in individual subclasses before they were analysed by gaschromatography. Hydrogenating the lipids makes it possible to circumvent in part difficulties which arise often with polyunsaturated fatty acids. As the chain length of C20 and C22 are mainly represented by C20:4 , the arachidonic acid and C22:6 the docosahexaenoic acid, both fatty acids can be assessed after hydrogenation. The fatty acid profile of phosphatidylcholine and phophatidylethanolamine of cardiac muscle from rat, guinea pig and pig was determined. Each sample was analysed before and after hydrogenation. The fatty acids with the same chain length were summed up and compared to the corresponding chain length after hydrogenation.     

Physical Properties of Butter Oil Fractions Obtained by Supercritical Carbon Dioxide Extraction

Butter oil has been fractionated with supercritical carbon dioxide at 40°C. 125 bar and 350 bar, respectively. The chemical composition and physical properties of the butter oil fractions have been studied with gas chromatography and differential scanning calorimetry (DSC). The polymorphism of the fractions has been studied by X-ray diffraction during crystallization. Fractionation carried out at lower extraction pressure results in higher selectivity. The first fractions collected during the extraction process contain triglycerides which are enriched in short chain fatty acids. The colourless fractions melt and crystallize at a lower temperature compared to the original butter. The residual fraction is enriched in triglycerides with long chain fatty acids, is yellow, melts and crystallizes at a higher temperature. All the investigated fractions shows the β′ form when crystallized from the melt.     

Detection of trace fatty acids in fats and oils by urea fractionation and gas-liquid chromatography

The detection of trace fatty acids (<0.1%) in a fat or oil by gas-liquid chromatography is possible when the methyl esters are fractionated with urea to provide a number of less complex fractions. Identification and estimation of trace fatty acids is simplified by quantitative removal of other fatty acids having similar gas chromatographic retention times. A detailed knowledge of the order in which inclusion compounds are formed was obtained by fractionating a complex mixture of marine and vegetable fatty acids. In addition, lanolin was fractionated to determine the preferential order in which saturated, branched chain (iso-, anteiso-) and hydroxy acids form inclusion compounds. Using urea fractionation and gas chromatography, 52 trace fatty acids were tentatively identified in butter, 30 in lard, and 26 in walnut oil. Presented at the AOCS Meeting in New Orleans, 1964.     

Rapid Analysis of Acylglycerols in Low Molecular Weight Milk Fat Fractions

A suitable analytical method was required to facilitate development of an industrial-scale short-path distillation (SPD) process. Short-path distillation produces milk fat distillates (MFD) enriched in low molecular weight milk fat components—viz. free fatty acids, monoacylglycerols, diacylglycerols, cholesterol and low molecular weight triacylglycerols. In this case, solid-phase extraction (SPE) was considered a better alternative than thin-layer chromatography for separating polar and apolar lipid components in MFD samples due to its speed and near-complete recoveries. Solid-phase extraction of MFDs yielded two fractions, both of which are sufficiently pure for subsequent analysis by gas chromatography. This procedure provided rapid and complete chemical characterization (including mass balances) of low-molecular weight milk-fat fractions.     

Lipids in sea water

Acidified and filtered sea water samples which were extracted with petroleum ether and ethyl acetate have been shown to contain a variety of lipid compounds in trace amounts. Concentrations of these solvent-soluble substances ranged from 0.5 to 6.0 mg/liter, the lower concentrations being found in offshore waters. The solvent extracts of the sea water were separated into eight lipid classes by column chromatography on silicic acid. The fractions eluted with solvents of increasing polarity were characterized by thin-layer chromatography, infrared and ultraviolet absorption and gas chromatography. These techniques revealed a complex mixture of alkanes, alkenes, fatty acids, steroids, phospholipids and many as yet unidentified components. Twenty to thirty alkanes were present as indicated by gas chromatography. No aromatic hydrocarbons were detected. Chromatography of the methyl esters of the fatty acids indicated the presence of acids with chain lengths varying from 14 to 22 carbons, both saturated and unsaturated. In many samples the unsaturated fatty acids containing 18 to 22 carbons predominated. The lipid components varied somewhat in composition as well as concentration from location to location and with season and depth.     

Impact of Fabp1 Gene Ablation on Uptake and Degradation of Endocannabinoids in Mouse Hepatocytes

Liver fatty‐acid‐binding protein (FABP1, L‐FABP) is the major cytosolic binding/chaperone protein for both precursor arachidonic acid (ARA) and the endocannabinoid (EC) products N‐arachidonoylethanolamine (AEA) and 2‐arachidonoylglycerol (2‐AG). Although FABP1 regulates hepatic uptake and metabolism of ARA, almost nothing is known regarding FABP1’s impact on AEA and 2‐AG uptake, intracellular distribution, and targeting of AEA and 2‐AG to degradative hepatic enzymes. In vitro assays revealed that FABP1 considerably enhanced monoacylglycerol lipase hydrolysis of 2‐AG but only modestly enhanced AEA hydrolysis by fatty‐acid amide hydrolase. Conversely, liquid chromatography–mass spectrometry of lipids from Fabp1 gene‐ablated (LKO) hepatocytes confirmed that loss of FABP1 markedly diminished hydrolysis of 2‐AG. Furthermore, the real‐time imaging of novel fluorescent NBD‐labeled probes (NBD‐AEA, NBD‐2‐AG, and NBD‐ARA) resolved FABP1’s impact on uptake vs intracellular targeting/hydrolysis. FABP1 bound NBD‐ARA with 2:1 stoichiometry analogous to ARA, but bound NBD‐2‐AG and NBD‐AEA with 1:1 stoichiometry—apparently at different sites in FABP1’s binding cavity. All three probes were taken up, but NBD‐2‐AG and NBD‐AEA were targeted to lipid droplets. LKO reduced the uptake of NBD‐ARA as expected, significantly enhanced that of NBD‐AEA, but had little effect on NBD‐2‐AG. These data indicated that FABP1 impacts hepatocyte EC levels by binding EC and differentially impacts their intracellular hydrolysis (2‐AG) and uptake (AEA).     

Relation of triglycerides to phosphoglycerides in fungi

Lipids of the fungiPhycomyces blakesleeanus, Lipomyces lipoferus, Glomerella cingulata andCoprinus comatus have been analyzed by physical and chemical methods. Triglycerides were the largest fraction of all the lipids in these fungi but significant amounts of phosphoglycerides were also present. The presence of relatively large amounts of triglycerides and phosphoglycerides, and the fatty acid patterns of these glycerides, suggests that formation of the tri- and phosphoglycerides involves participation of key intermediates from a common pathway of synthesis. The triglycerides ofGlomerella cingulata have been studied in more detail than those of the other species. It has been found, using preparative thin-layer chromatography and analytical gas-liquid chromatography, thatG. cingulata triglycerides comprise one fraction of saturated and monoenoic fatty acids, another fraction of saturated, mono-, and dienoic acids and two fractions containing varying proportions of saturated, mono-, di-, and trienoic fatty acids.     

Species difference of liver cytosolic fatty acid-binding protein in rat,mouse and guinea pig

Binding properties of liver cytosolic protein for oleic acid, palmitoyl-CoA and bromosulphophthalein (BSP) were compared for rat, mouse and guinea pig. Hepatic cytosol of rat, mouse and guinea pig contained proteins with a molecular weight of ca. 12,000 and had an affinity for [1-¹⁴C]-oleic acid. The concentration of fatty acid-binding protein (FABP) was almost the same in livers of the animals of the 3 species and was ca. 50 μg/mg cytosolic protein. Electrophoretic studies revealed that FABP from hepatic cytosol of rat, mouse and guinea pig, purified with affinity chromatography, are distinct from one another in terms of their charge. FABP of rat liver was capable of binding any 3 ligands-oleic acid, palmitoyl-CoA and BSP—at relatively high binding capacity. FABP of mouse liver also bound oleic acid and palmitoyl-CoA to a great extent, but its binding capacity for BSP was only one-third that of rat liver. FABP of guinea pig liver bound less oleic acid and palmitoyl-CoA than rat liver, whereas it had almost the same binding capacity for BSP as rat liver.     

Detection of 430 Fatty Acid Methyl Esters from a Transesterified Butter Sample

Milk fat is known to contain one of the highest number of fatty acids of all edible oils. Some of these fatty acids are known to be valuable (e.g. conjugated linoleic acids, furan fatty acid) and other as undesirable (e.g. saturated and some trans-fatty acids) food ingredients. However, a comprehensive picture on the presence of many trace fatty acids has not been achieved. For this reason we have developed an analysis scheme based on the conversion of the fatty acids into methyl esters. The fatty acid methyl esters were then fractionated by urea complexation. Both the filtrate of the urea complexation (~4 % of the sample weight) and the original sample were fractionated by high-speed counter-current chromatography (HSCCC). The resulting fractions were analyzed by GC/MS analysis. With this method 430 fatty acids were detected in one single butter sample. More than 230 fatty acids had two or more double bonds. In addition to the widely known spectrum of fatty acids we also detected a range of cyclohexyl fatty acids (five homologues) and methyl-branched fatty acids (including short chain and even-numbered anteiso-fatty acids), conjugated tetradecadienoic acids along with the novel ω-oxo-fatty acids (seven homologues). The reported relative retention time on the polar column may serve as a data base for the screening of other samples for this profusion of fatty acids.     

Rat hepatocyte plasma membrane acyl:CoA synthetase activity

The presence of long chain acyl:CoA synthetases in mammalian microsomes and mitochondria has been established previously by Aas (Biochim. Biophys. Acta 231, 32–47 [1971]). The presence of a plasma membrane-associated enzyme was investigated in rat hepatocyte plasma membranes, where an enzyme exhibiting high activity and with a preferred substrate of 18-carbon chain length was discovered. The results are consistent with the presence of a single enzyme. The effect of the degree of unsaturation of the fatty acid substrates was not as pronounced as that arising from the length of the carbon chain. The pattern of substrate preference of the enzyme was ω3 polyenoic fatty acids >ω6 polyenoic acids >ω9 monoenoic acids > saturated acids. This may relate to the similar substrate preference pattern exhibited by the fatty acyl desaturase enzymes. The role played by long chain acyl:CoA synthetase in hepatocyte metabolism is uncertain, but it may relate to the incorporation of polyenoic fatty acids from the circulation into cell membranes and the trapping of other fatty acids within the cell for further metabolism.     

Lipoide tierischer Organ- und Depotfette in Abhängigkeit von der Fütterung I: trans-Fettsäuren in Kälber- und Schweinefetten

Dietary Fat and the trans-Unsaturated Fatty Acids in Bovine and Porcine Tissues of Heart, Liver and Kidney and Depot Fats from different Localities within the same Animal The content of trans-unsaturated fatty acids in lipids of heart, liver and kidney and depot fats from different localities within the same animal (visceral, pericardial, perinephric, extrahepatic, subcutaneous and/or back fat and fat attached to ribs) of calves and pigs, fed a basal diet without trans-unsaturated fatty acids or fed a hardened fat diet, was determined by IR-spectrography. The lipids of heart, kidney and liver were separated by Silica Gel G column chromatography in cholesterol ester, neutral lipids and phospholipid fractions. The trans-unsaturated fatty acids of each were determined.