Studies on the compartmentation of lipid in adipose cells. I. Subcellular distribution, composition, and transport of newly synthesized lipid: liposomes

The subcellular distribution and composition of endogenously synthesized lipid in isolated white adipose cells were studied to determine the nature and extent of lipid compartmentation. After brief incubation of cells with labeled glucose, acetate, or palmitic acid, over 90% of newly synthesized triglyceride was localized in the bulk-lipid phase, indicating rapid intracellular transport and storage. From 13 to 20% of the newly formed lipid was diglyceride, and over 95% of it was localized in the central lipid-storage vacuole rather than in organelle systems concerned with esterification, thus indicating intracellular segregation of newly synthesized partial glycerides. Most of the newly synthesized phosphatides partitioned with membranous organelles. Synthesis of cholesterol or cholesteryl ester was negligible. After brief incubation of cells with labeled glucose, the relative specific activity of organelle triglyceride was mitochondria > microsomes > liposomes > soluble supernatant > bulk lipid. In pulse-chase studies the specific activity of organelle triglyceride decreased and that of the bulk fraction increased reflecting intracellular lipid transport. The data suggest that a significant proportion of newly formed lipid is transferred from mitochondrial membranes into the storage vacuole by direct lipid-lipid interaction. Liposomes, which consist of small enclosed lipid droplets resembling chylomicrons, contained triglycerides of specific activity similar to microsomal triglyceride. While the evidence that liposome triglyceride may be microsomal in origin is indirect, the results do indicate that the liposome fraction represents a phase in the transport and(or) storage of new glyceride. At least two forms of compartmentation of newly synthesized lipids occurred. The first, termed "structural," refers to localization of lipids to organelle fractions. The second type of compartmentation, termed "chemical," concerns the intracellular segregation of a specific lipid class. The accumulation and segregation of newly synthesized diglyceride in the bulk storage pool are examples of the latter form of compartmentation.     

A favourable assessment of the O.P.T.--histamine reaction in the method of von Redlich and Glick

During the course of an investigation of the properties of triglyceride lipase in nerve endings of the central nervous system (1) there arose a need for rapid determinations of a lipase in a large number of membrane preparations. This communication reports a procedure for the determination of glyceride lipase in which fatty acid, formed from a radioactive substrate by the lipase, is separated from glycerides on DEAE-cellulose paper.     

Concentrations of glycerides and phospholipids in rat heart and gastrocnemius muscles: Effects of alloxan-diabetes and perfusion

1. Methods are described for the extraction of lipid and assay of mono-, di- and tri-glyceride glycerol and phospholipid phosphorus in rat heart and gastrocnemius muscles. 2. In hearts from normal animals, concentrations found were: monoglyceride, 0·6; diglyceride, 0·1; triglyceride, 12·6μmoles of glyceride glycerol/g. of dry muscle; phospholipid, 171μg.atoms of phospholipid phosphorus/g. of dry muscle. Concentrations of glycerides in gastrocnemius muscle were similar to heart muscle but those of phospholipids were lower (64μg.atoms of phospholipid phosphorus/g. of dry muscle). 3. Alloxan-diabetes increased the concentration of triglyceride in the muscles twofold. This increase was shown to be dependent in the heart on the availability of growth hormone and cortisol but not on the availability of dietary lipid. Total glyceride in the heart was increased after 48 and 72hr. starvation but not after 96hr. Changes in glyceride concentration seen in starvation and diabetes were not associated with significant changes in phospholipid concentration. It is suggested that mobilization of free fatty acids in diabetes leads to the synthesis of additional glyceride in muscle. 4. The possible contribution of glyceride fatty acid in the heart to respiration during perfusion has been calculated from the net loss of glyceride during perfusion, and also from the relative rates of lipolysis and esterification and compared with oxidation of fatty acid required for the balance of oxygen consumption (oxygen not utilized in the oxidation of glucose or glycogen glucose). In the normal or diabetic heart perfused with glucose and insulin the breakdown of glyceride can account for the balance of oxygen consumption. In the normal heart perfused without substrate the balance of oxygen consumption is not entirely accounted for by the breakdown of glyceride.     

Hydrolysis of primary and secondary esters of glycerol by pancreatic juice

The relative rates of hydrolysis of the secondary ester in glycerol 1,3-benzylidene 2-oleate and in glycerol 1,3-dihexadecyl ether 2-oleate, and of the primary and secondary esters in triolein were determined. Both unaltered and selectively inactivated rat pancreatic juice were used as sources of enzyme. It was found that rat pancreatic juice contains an enzyme that can hydrolyze fatty acids esterified at the 2-position of a glyceride. This enzyme is not pancreatic lipase. It may be sterol ester hydrolase. Partial glycerides, as well as complete glycerides, can serve as substrates. Pancreatic lipase, if it can hydrolyze the 2-positioned fatty acids of a triglyceride, does so at a very slow rate.     

Retention of lipolytic products in chylomicrons incubated with lipoprotein lipase: electron microscope study.

Early effects of lipolysis on the structure of chylomicrons in vitro were studied in rat chylomicrons incubated with purified bovine mild lipoprotein lipase at pH 8.1. The amount of the albumin added to the incubation medium was limited so that free fatty acids (FFA) and partial glycerides formed during lipolysis would accumulate in the chylomicrons. The structures visualized in lipolyzed chylomicrons was found to be affected by pH during preparation of specimens for microscopy, whether fixed with OsO4 and sectioned, or stained with sodium phosphotungstate and examined as whole mounts. Circular aqueous spaces were present in the triglyceride core of lipolyzed chylomicrons processed at pH 8.1 and 7.4. Sometimes the spaces contained aggregates of osmiophilic material and whorls of bilayered lamellae. The spaces were replaced by lamellar structures having a periodicity of 40 A, in chylomicrons processed at pH 5.5, and the spaces and lamellae were both absent at pH 3.0. The findings indicate that these spaces were lined by a lipid monolayer which formed bilayered lamellae under certain conditions. It is concluded that the monolayer lining the aqueous spaces is an inward extension of the chylomicron surface film produced by the accumulation and movement of lipolytic products, FFA and partial glycerides, in the interfacial plane between core triglyceride and water.     

The viscosity behavior and conformations of low-molecular-weight poly-γ-benzyl-L-glutamate in various solvents

Reduced viscosity and infrared spectra of low-molecular-weight poly-γ-benzyl-L -glutamate (which was prepared by polymerization of the N-carboxyanhydride with n-hexylamine initiation at [A]/[I] 3, 4, and 8) have been measured in various organic solvents. Infrared spectra indicate that the polypeptide molecules consist of a series of residues of two forms, the solvated σ-form and the hydrogen-bonded β-form, and relative abundance of the two forms depends on solvent species and polypeptide concentration. An approximate method is developed for estimating the content of β-structure from a single spectrum of dissolved polypeptide. The reduced viscosity of some solutions is scarcely dependent in polypeptide concentration, in which a single conformation is predominantly kept over the concentration range. In the other solutions the reduced viscosity displays a strong concentration dependence or some anomalous behavior. The observed viscosity behavior has been attributed to the changes in size and shape of aggregates, which are determined by the number of hydrogen bonds in the aggregate. This unusual behavior is exhibited by solutions of the polypeptides which have a moderate content of β-structure at a finite concentration. Both the content of β-structure and the extent of association increase in the following solvents, ranked in order of effectiveness: dimethylformamide, trifluoroethanol < trimethyl phosphate < chloroform < dioxane < ethylene dichloride < ethylene dibromide. Infrared spectra suggest that the conformation of the polypeptide in dichloroacetic acid differs from either the σ- or the β-conformation.     

Adipose tissue glyceride synthesis in patients with hyperapobetalipoproteinemia

Adipose tissue was obtained at thoracotomy in five control patients with valvular heart disease, all of whom were free of coronary artery disease and all of whom were normolipidemic with normal low density lipoprotein apolipoprotein B levels, and eight patients with coronary artery disease, all of whom had hyperapobetalipoproteinemia. In both groups, the rates at which linoleic acid and palmitic acid were incorporated into diglyceride and triglyceride were determined in vitro. The data indicate that fatty acid incorporation into adipose tissue glycerides was twice as rapid in controls as in patients with hyperapobetalipoproteinemia. By contrast there was no difference between the groups in the rate of net lipolysis of adipocyte glyceride. The data at hand do not establish the mechanism responsible for the difference in synthesis between normal subjects and patients with hyperapobetalipoproteinemia, but this may explain the delayed chylomicron triglyceride clearance previously observed in the disorder.     

Characterization and Biological Activity of the Monocytosis-producing Agent of Listeria monocytogenes

Experiments directed toward determining the lipids in extracts of Listeria monocytogenes containing monocytosis-producing agent (MPA) and the effect of these extracts on several biochemical parameters previously shown to change during experimental Listeria infection were conducted. MPA-containing extracts were found to be a complex of lipids with glycerides, glycolipids, and phospholipid being present. No common cell wall carbohydrates were found. A glyceride, designated glyceride A, was determined to cause the characteristic mononuclear response observed in mice injected with MPA-containing extracts. Fasted MPA-treated animals showed less gluconeogenesis than did controls. Blood glucose levels declined in MPA-treated animals. Increases observed in both blood urea nitrogen and plasma glutamic-pyruvic transaminase were greater in the control groups. Incorporation of (14)C-alanine into liver glycogen was depressed in MPA-treated animals. Liver steroid levels in the control groups increased during fasting and remained elevated for the duration of the experiments, while levels in the MPA-treated groups declined initially and showed no increase until 72 hr after injection. MPA appears to affect steroid metabolism and consequently the animals' homeostatic mechanisms seem to be impaired. Possibly as a consequence, carbohydrate metabolism is altered. The apparent effect of MPA on steroid metabolism and on the gluconeogenic process may indicate participation in the carbohydrate derangement observed in experimental Listeria infection.     

Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media

Several surfactant-coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C(18)Delta(9)GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant-coated lipase was carried out in organic media. The surfactant-coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant-coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di-substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. (c) 1995 John Wiley & Sons, Inc.     

Rapid effects of insulin and glucose on the hepatic incorporation of gluconeogenic substrates into glyceride glycerol and glycogen

1. The hepatic utilization of gluconeogenic substrates was investigated shortly after portal infusion of either insulin or glucose in fasted rats. 2. After 20 min of insulin infusion blood glucose concentration decreased. However, neither glucose generation from precursors such as alanine or pyruvate nor their incorporation into fatty acids was modified. Under these conditions, insulin rapidly increased the incorporation of gluconeogenic substrates into the hepatic glyceride glycerol fraction. Insulin treatment led to a decrease in substrate incorporation into liver glycogen. 3. After 20 min of portal glucose infusion both plasma insulin and glucose concentrations increased and the incorporation of pyruvate into hepatic glyceride glycerol and into glycogen was also stimulated. 4. A close relationship was observed between blood glucose concentrations and the level of incorporation of gluconeogenic substrates into liver glycogen. 5. In conclusion, during fasting insulin stimulates the incorporation of gluconeogenic substrates into the glycerol moiety of hepatic glycerides, which may be the preferential mechanism through which fatty acid esterification is accomplished during refeeding. This effect of insulin is rapid and detected even before other classical modifications induced by the hormone such as gluconeogenesis inhibition or lipogenesis activation. Furthermore, the effect is not related to insulin-induced hypoglycemia since glucose infusion mimics insulin action on glyceride glycerol synthesis.     

A new approach to determine the stereospecificity in lipase catalysed hydrolysis using circular dichroism (CD): lipases produce optically active diglycerides from achiral triglycerides

We describe a sensitive CD method for determining the stereospecificity in lipase (E.C.3.1.1.3) catalysed hydrolysis of triacyl glycerols into diacyl glycerols. The diglycerols were converted to chiral tert-butyldimethylsilylated 1,2- or 2,3-di-O-benzoyl-sn-glycerol (5 or 5'), and their CD was measured. This approach showed for the first time that lipases produce optically active diacyl glycerides from achiral tripalmitin and tribenzoyl glyceride with a variable extent of enantioselectivity depending on the acyl groups and the enzymes.     

Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

Pseudomonas lipase immobilized on CaCO₃ powder was used for the glycerolysis of n−3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO₃ powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.     

Stability of a liposomal formulation containing lipoyl or dihydrolipoyl acylglycerides

Context: The acylglycerides of lipoic and dihydrolipoic acids may serve as slow-release sources for cutaneous delivery of these antioxidants when formulated in a liposomal vehicle.

Objective: Testing was conducted to determine the storage stability of lipoyl glycerides in phospholipid-based liposomes.

Materials and methods: Lipoyl glycerides prepared by transesterification of lipoic acid with high oleic sunflower oil were incorporated into unilamellar liposomes comprised of soy phosphatidylcholine (soyPC) or dioleoylphosphatidylcholine (DOPC).

Results: Lipoyl glycerides were stable in soyPC at 4?°C (90% remaining after five weeks) and decayed with a half-life (t_½) of 14?d at 40?°C. In contrast, lipoyl glycerides embedded in DOPC were completely stable for four weeks at 40?°C. Dihydrolipoyl glycerides in soyPC converted to lipoyl glycerides at 4?°C (t_½?=?14?d) over four weeks, and much more rapidly so at 40?°C (t_½?=?1?d). A hydroperoxide accumulation analysis indicated that lipoyl glycerides and dihydrolipoyl glycerides were modified or degraded while suppressing autoxidation of the polyunsaturated fatty acids present in soyPC. Dynamic light scattering measurements found that liposomes containing lipoyl glycerides or dihydrolipoyl glycerides did not undergo significant size changes for at least 48?d, indicating that inclusion of the lipoic acid derivatives did not induce vesicle aggregation.

Discussion/Conclusion: Substitution of the soyPC with DOPC, which is not readily subject to autoxidation, provided a much more stable storage environment for lipoyl glycerides. These findings confirm the expectation that phospholipid liposomes need to be oxidatively stable vehicles for dermal delivery of lipoic acid derivatives.     

Pluronic and Tetronic Copolymers with Polyglycolyzed Oils as Self-Emulsifying Drug Delivery Systems

The potential of poly(ethylene oxide)-poly(propylene oxide) block copolymers Pluronic F127 (PF127) and Tetronic 304 (T304), 904 (T904) and 1307 (T1307) as components of solid self-(micro)emulsifying dosage forms, S(M)EDDS, was evaluated. The dependence of the self-associative properties of Tetronics on pH explained the low ability of the micelles to solubilize griseofulvin at acid pH (sevenfold increase) compared to at alkaline pH (12-fold). Blends of polyglycolyzed glycerides (Labrasol, Labrafac CC, and Labrafil M 1944CS) with each copolymer at two different weight ratios (80:20 and 60:40) were prepared, diluted in water, and characterized in terms of globule size, appearance and griseofulvin solubility. The blends with Labrasol led to microemulsions that are able to increase drug solubility up to 30-fold. SMEDD hard gelatine capsules filled with griseofulvin and Labrasol or Labrasol/copolymer 80:20 showed a remarkable increase in drug solubility and dissolution rate, particularly when T904, T1307 or PF127 was present in the blend. This effect was more remarkable when the volume of the dissolution medium was 200 ml (compared to 900 ml), which can be related to a higher stability of the microemulsion when there is a greater concentration of the copolymer and glyceride in the medium.     

Chloroplastic Glyceride Isoform of Dihydroxyacetone Phosphate Reductase from Dunaliella tertiolecta: Purification and Characterization

The chloroplastic glyceride isoform of dihydroxyacetone phosphate reductase (Gly-DHAPR) in the photosynthetic unicellular green algae, Dunaliella, plays key role in the synthesis of glycerol-P and glycerides. A four-step procedure has been developed to purify the Gly-DHAPR from the chloroplasts of Dunaliella tertiolecta. The enzyme was purified 462-fold to apparent electrophoretic homogeneity by precipitation of Rubisco by polyethylene glycol-4000, and successive chromatography on DEAE cellulose, Sephacryl S-200, and Red Agarose. The overall yield of the purified enzyme was 5.1% with a specific activity of 425 μmol. min^?1. mg^?1 protein, and a subunit molecular mass of 37 kD. The Gly-DHAPR had little preference for NADH or NADPH, but was highly specific for DHAP. The purified enzyme was slightly stimulated by 50 mM NaCl, KCl or by 25 mM MgCl₂. Detergents, lipids, fatty acids, or long-chain acyl-CoA derivatives inhibited the Gly-DHAPR. The Gly-DHAPR differs in properties from the other chloroplastic osmoregulatory isoform of DHAP reductase from Dunaliella, but has significant similarities with the glyceride isoforms from higher plants for glycerol-P and triglyceride synthesis.     

Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-<Emphasis Type="Italic">epi</Emphasis>-5-<Emphasis Type="Italic">epi</Emphasis>-valiolone synthase gene in <Emphasis Type="Italic">Streptomyces albus</Emphasis> ATCC 21838

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.     

The photostability of different chlorophyll forms in dark grown leaves of wheat

Formulae were developed for calculation of the relative amount of different pigment forms of dark grown leaves of wheat, present before and after photoreduction of the protochlorophyllide. Three pigment forms were calculated from in vivo absorption spectra: the photoreducible protochlorophyllide with absorption maximum at 650 nm and the two chlorophyll(ide) forms with absorption maximum at 684 nm and 673 nm, respectively. The formulae were used to study the changes of the pigment forms at repeated photoreduction of the protochlorophyllide, and at a repeated treatment involving photoreduction of the protochlorophyllide followed by partial photo-decomposition of the chlorophyllide formed. Five consecutive photoreductions and reaccumulations of protochlorophyllide were carried out by high intensity irradiations of one second (red light, 700 W m^-2) given at intervals of 3 h. The results show that the pool size of reaccumulated protochlorophyllide decreased sharply with the number of photoreductions performed. The absorption spectrum of the chlorophyllide formed at each photoreduction proceeded through the Shibata shift (transformation of the 684-form to the 673-form) and the late red-shift (transformation of the 673-form to other pigment form(s) in the dark). High intensity irradiation for ten minutes (red light, 700 W m^-2) immediately after each phototransformation caused a photodecomposition of about three quarters of the newly formed chlorophyllide (which was in the 684-form) while the earlier formed chlorophyll(ide) (in the 673-form) appeared not to be decomposed. This partial photodecomposition of the chlorophyllide had no effect on further accumulation of protochlorophyllide in the dark, and the absorption spectrum of the remaining chlorophyllide proceeded through the Shibata shift. The partial photodecomposition caused an inhibition of the late red-shift, and the accumulated chlorophyll(ide) remained in the 673-form.     

Synthesis of S-adenosylmethionine synthetase isozymes from rat and mouse livers: Immunochemical studies

The α- and β-forms of S-adenosylmethionine synthetase in rat liver were completely fractionated by chromatography on a hydrophobic resin, phenyl-Sepharose. The α-form was eluted in low-ionic strength buffer, and the β-form was eluted with 50% dimethylsulfoxide. The α-form is less sensitive to dimethylsulfoxide, whereas the β-form is strikingly stimulated by dimethylsulfoxide, after removal of the dimethylsulfoxide. The levels of the α-form activity in rat liver after treatment with ethionine and adenine for 2 consecutive days, and those of the β-form activity in mouse liver on the 12th day after transplantation of Ehrlich ascites tumor cells, were increased several fold compared to normal liver. Immunochemical titrations with specific antibody against the β-form as well as kinetic studies indicated that the observed increase in the levels of each activity from the S-adenosylmethionine synthetase isozymes is due to an increase in the cellular content of the enzyme.     

Expression of a constitutively active insulin receptor in Drosulfakinin (Dsk) neurons regulates metabolism and sleep in Drosophila

The ability of organisms to sense their nutritional environment and adjust their behavior accordingly is critical for survival. Insulin-like peptides (ilps) play major roles in controlling behavior and metabolism; however, the tissues and cells that insulin acts on to regulate these processes are not fully understood. In the fruit fly, Drosophila melanogaster, insulin signaling has been shown to function in the fat body to regulate lipid storage, but whether ilps act on the fly brain to regulate nutrient storage is not known. In this study, we manipulate insulin signaling in defined populations of neurons in Drosophila and measure glycogen and triglyceride storage. Expressing a constitutively active form of the insulin receptor (dInR) in the insulin-producing cells had no effect on glycogen or triglyceride levels. However, activating insulin signaling in the Drosulfakinin (Dsk)-producing neurons led to triglyceride accumulation and increased food consumption. The expression of ilp2, ilp3 and ilp5 was increased in flies with activated insulin signaling in the Dsk neurons, which along with the feeding phenotype, may cause the triglyceride storage phenotypes observed in these flies. In addition, expressing a constitutively active dInR in Dsk neurons resulted in decreased sleep in the fed state and less starvation-induced sleep suppression suggesting a role for insulin signaling in regulating nutrient-responsive behaviors. Together, these data support a role for insulin signaling in the Dsk-producing neurons for regulating behavior and maintaining metabolic homeostasis.     

Glyceride Structure and Biosynthesis of Natural Fats

In order to study specificity of pancreatic lipase, a number of synthetic triglycerides were hydrolyzed by the enzyme under an improved condition. The proportions of isomers of the derived mono- and diglycerides, and the fatty acid compositions of the derived free acids and monoglycerides were determined. The hydrolyzing rate of fatty acids in glycerides depended on the position esterified in the glycerol, carbon number of the acid, and structure of the glyceride. Positional specificity of the enzyme was markedly displayed for symmetrical triglyceridcs composed of long chain acids, but at somewhat lower rate for glycerides containing short chain or highly unsaturated acids.