Stimulation of the production of unesterified fatty acids in nerve endings of guinea-pig brain in vitro by noradrenaline and 5-hydroxytryptamine

1. Noradrenaline (1mm) and 5-hydroxytryptamine (1mm) stimulated the production of unesterified palmitate, oleate, stearate and arachidonate in nerve endings (synaptosomes) isolated from combined guinea-pig cerebral cortex and cerebellum. 2. Iproniazid phosphate (0.36mm) increased the concentrations of the same acids in osmotically ruptured synaptosomes. Further addition of 1mm-noradrenaline or 1mm-5-hydroxytryptamine reversed this increase. 3. Noradrenaline (0.01mm) stimulated the production of unesterified fatty acids in isolated synaptic membranes. 5-Hydroxytryptamine (0.01mm) stimulated the production of unesterified fatty acids in synaptic membranes and synaptic vesicles.     

Monoglyceride and diglyceride lipases from human platelet microsomes

In the present study, we have characterized the properties of both diglyceride lipase (lipoprotein lipase, EC 3.1.1.24) and monoglyceride lipases (acylglycerol lipase, EC 3.1.1.23) in an attempt to assess the potential roles of these two enzymes in the release of arachidonate in activated human platelets. Diglyceride lipase exhibited maximal activity at pH 3.5, whereas monoglyceride lipase showed optimal activity at pH 7.0. Neither of the lipases were inhibited by EDTA or stimulated by Ca2+, Mg2+ or Mn2+. Both enzymes, however, were strongly inhibited by Hg2+ and Cu2+, indicating the involvement of sulfhydryl groups in catalytic activity. This suggestion was further supported by their sensitivity toward sulfhydryl inhibitors, with monoglyceride lipase being more susceptible to inhibition. Both lipases were found to be inhibited to a different degree by a variety of antiplatelet drugs blocking aggregation and arachidonate release. Kinetic studies indicated that dichotomous metabolism of diacylglycerol to monoacylglycerol and to phosphatidic acid could occur concurrently, since the apparent Km values for diglyceride lipase and for diglyceride kinase were comparable. Further studies showed that the specific activity of monoglyceride lipase was at least 100-fold higher than that of diglyceride lipase, indicating that the rate-limiting step in the release of arachidonate was the reaction catalyzed by diglyceride lipase.     

The stimulation by transmitter substances and putative transmitter substances of the net activity of phospholipase A2 of synaptic membranes of cortex of guinea-pig brain.

1. The distribution of the hydrolyses of phosphatidylcholine by phospholipase A2 and phospholipase A1, and the hydrolysis of lysophosphatidylcholine by lysophospholipase, in subcellular and subsynaptosomal fractions of cerebral cortices of guinea-pig brain, was determined. 2. Noradrenaline stimulated hydrolysis by phospholipase A2 in whole synaptosomes, synaptic membranes and fractions containing synaptic vesicles. 3. Stimulation of hydrolysis by phospholipase A2 in synaptic membranes by noradrenaline was enhanced by CaCl2, and by a mixture of ATP and MgCl2. The optimum concentration of CaCl2, in the presence of ATP and MgCl2, for stimulation by 10 muM-noradrenaline was in the range 1-10muM. The optimum concentration for ATP-2MgCl2 in the presence of 1 muM-CaCl2 was in the range 0.1-1mM. 4. Hydrolysis by phospholipase A2 of synaptic membranes was also stimulated by acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine), histamine, psi-aminobutyric acid, glutamic acid and aspartic acid. With appropriate concentrations of cofactors, sigmoidal dose-response curves were obtained, half-maximum stimulations being obtained with concentrations of stimulant in the range 0.1-1muM. 5. Taurine also stimulated hydrolysis of phosphatidylcholine by phospholipase A2. There were only slight stimulations with methylamine, ethylenediamine or spermidine. No stimulation was obtained with glucagon.     

Release of arachidonate from diglyceride in human platelets requires the sequential action of a diglyceride lipase and a monoglyceride lipase

Release of arachidonate from 2-arachidonyl diglyceride by human platelet microsomes was investigated. Diglycerides labeled with ¹⁴C-stearate at sn-1 and with ³H-arachidonate at sn-2 were used as a substrate for microsomal diglyceride lipase. Diglyceride was deacylated first at sn-1 as evidenced by the accumulation of 2-arachidonyl monoglyceride but not of 1-stearoyl monoglyceride. Subsequent release of arachidonate from monoglyceride required the action of a monoglyceride lipase. Studies on substrate specificity indicated that diglyceride lipase utilized 2-arachidonyl diglyceride as the best substrate.     

Enzyme activities and isozyme composition of triglyceride,diglyceride and monoglyceride lipases in Periplaneta americana,Locusta migratoria and Polia adjuncta

• 1.1. Measurements of maximal enzyme activities were combined with an electrophoretic study of isozyme make-up in an examination of triglyceride, diglyceride and monoglyceride lipases from the flight muscle, fat body and gut of the cockroach, Periplaneta americana and the locust, Locusta migratoria and from the flight muscle and fat body of the moth, Polia adjuncta.
• 2.2. Tri-, di- and mono-glyceride lipases were present in all tissues of the insects with diglyceride lipase ≥ triglyceride lipase activity in all cases and monoglyceride lipase ≥ diglyceride lipase activity in locust and moth.
• 3.3. In the flight muscle, a strong correlation was found between the activities of lipases and the known use of lipid as a fuel for flight in these insects. Lipase activities were lowest in the cockroach (a carbohydrate-based flight metabolism), intermediate in the locust (both carbohydrate and lipid-fueled flight), and highest in the moth (a non-feeding, lipid-catabolizing adult) flight muscle.
• 4.4. Polyacrylamide gel electrophoresis, using substrate-impregnated gels and stained for fatty acids released by lipase action, demonstrated the presence of tissue specific isozymes of tri-, di- and mono-glyceride lipases in the three insects. In addition, some, but not all, tissues showed multiple molecular forms of one or more of the lipases.
• 5.5. Diglyceride and monoglyceride lipase activities in both flight muscle and fat body of the insects coelectrophoresed suggesting the possibility that these two lipase activities might be catalyzed by a single enzyme protein.

     

The stimulation by synaptic transmitters of the incorporation of oleate into the phospholipid of synaptic membranes.

Noradrenaline stimulated the incorporation of oleate into choline glycerophospholipids of guinea-pig brain synaptic membranes incubated in sodium phosphate buffer. In the presence of 1 mm-NaF, noradrenaline stimulated the incorporation of oleate into the choline glycerophospholipids, phosphatidylinositol, ethanolamine glycerophospholipids, phosphatidylserine and phosphatidic acid of synaptic membranes incubated in 10 mm-Tris-HCl buffer. In Tris-CHl containing 1 mm-NaF, stimulation of incorporation of oleate into choline glycerophospholipids by noradrenaline was enhanced by ATP, CaCl2, MgCl2 and CoA plus dithiothreitol. The optimum concentration of CaCl2 for stimulation by 10 mum-noradrenaline was 10 mum. In the presence of CaCl2, the optimum concentration of ATP-2MgCl2 was in the range 0.1-1 mm. Acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine, histamine and gamma-aminobutyric acid also stimulated the incorporation of oleate into choline glycerophospholipids of synaptic membranes. Sigmoidal dose-response curves were obtained, similar to those obtained previously for stimulation by the same agonists of the hydrolysis of phosphatidylcholine by phospholipase A2 (Gullis & Rowe, 1975a). The initial rate of transfer of oleate from oleoyl-CoA to choline glycerophospholipid was similar to the initial rate of transfer from oleate-albumin, stimulated by noradrenaline. Transfer of oleate from oleoyl-CoA was not appreciably stimulated by noradrenaline, but was stimulated by ATP and MgCl2.     

Haemolymph lipids and fat body lipases of the southern armyworm moth

The haemolymph lipid of the southern armyworm moth, Prodenia eridania, is chiefly diglyceride with smaller amounts of triglyceride, monoglyceride, and free fatty acid also present. The stored lipid of moth fat body is almost all triglyceride. Although flight muscle contains a very active monoglyceride lipase, its ability to hydrolyse tri- and diglycerides is very low. The fat body contains enzymes able to hydrolyse tri-, di-, and monoglycerides. These data do not support the suggestion that fat body triglyceride is converted to diglyceride, which is carried in the haemolymph to the flight muscle and then hydrolysed to free fatty acid for oxidation during flight; rather, they indicate that triglyceride can be completely hydrolysed in the fat body, and the resulting free fatty acid is carried to the flight muscle to provide energy for flight.     

Studies on the hormone-sensitive lipase of adipose tissue

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.     

Growth of Acinetobacter sp. strain HO1-N on n-hexadecanol: physiological and ultrastructural characteristics.

The growth of Acinetobacter sp. strain HO1-N on hexadecanol results in the formation of intracytoplasmic membranes and intracellular rectangular inclusions containing one of the end products of hexadecanol metabolism, hexadecyl palmitate. The intracellular inclusions were purified and characterized as "wax ester inclusions" consisting of 85.6% hexadecyl palmitate, 4.8% hexadecanol, and 9.6% phospholipid, with a phospholipid-to-protein ratio of 0.42 mumol of lipid phosphate per mg of inclusion protein. The cellular lipids consisted of 69.8% hexadecyl palmitate, 22.8% phospholipid, 1.9% triglyceride, 4.7% mono- and diglyceride, 0.1% free fatty acid, and 0.8% hexadecanol, as compared with 98% hexadecyl palmitate and 1.9% triglyceride, which comprised the extracellular lipids. Cell-associated hexadecanol represented 0.05% of the exogenously supplied hexadecanol, with hexadecyl palmitate accounting for 14.7% of the total cellular dry weight. Acinetobacter sp. strain HO1-N possesses a mechanism for the intracellular packaging of hexadecyl palmitate in wax ester inclusions, which differ in structure and chemical composition from "hydrocarbon inclusions" isolated from hexadecane-grown cells.     

Appearance of monoglyceride and triglyceride in the cell envelope of Escherichia coli mutants defective in diglyceride kinase

Diglyceride kinase mutants of Escherichia coli contain about 50- to 100-fold more 1,2-diglyceride than wild type cells. We now report that monoglyceride and triglyceride also accumulate in these strains. In mutant RZ60 (dgk-6) these compounds represent about 1 and 0.2%, respectively, of the total lipid fraction, while diglyceride represents 5-8% under most conditions. Monoglyceride accumulates predominantly in the outer membrane, while triglyceride builds up together with diglyceride in the cytoplasmic membrane. Under typical growth conditions about two-thirds of the diglyceride in E. coli arises in conjunction with synthesis of the membrane-derived oligosaccharides (Raetz, C.R.H., and Newman, K.F. (1979) J. Bacteriol. 137, 860-868). Inhibition of membrane-derived oligosaccharides (MDO) synthesis also curtails the accumulation of monoglyceride and triglyceride. However, there appears to be at least one other MDO-independent source of diglyceride and related metabolites. Since MDO synthesis is suppressed by high osmolarity (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S. A. 79, 1092-1095), we have examined the effects of osmolarity on diglyceride accumulation in RZ60 (dgk-6). As expected, if MDO synthesis and diglyceride formation are coupled, the diglyceride level in RZ60 is higher at low osmolarity, while at high osmolarity the level of diglyceride is reduced to that observed in double mutants defective both in MDO synthesis and diglyceride kinase. Since dgk mutants do not grow at very low osmolarity, we have isolated several spontaneous phenotypic revertants that do. One class regains diglyceride kinase and has low diglyceride levels under all conditions. The other class remains defective in diglyceride kinase but tolerates higher diglyceride levels which amount to 13% of the total lipid during maximal induction of MDO synthesis at low osmolarity.     

RHC 80267 does not inhibit the diglyceride lipase pathway in intact platelets

RHC 80267 inhibits diglyceride lipase activity in microsomes from canine platelets (1). Chau and Tai (2) reported that RHC 80267 prevents the transient accumulation of monoglyceride in thrombin-stimulated human platelets, while leaving arachidonate release unimpaired. In contrast, we find that while the drug inhibits both diglyceride lipase (I₅₀=15 μM) and monoglyceride lipase (I₅₀=11 μM) activities in platelet microsomes, it is ineffective when added to intact platelets. The transient intermediates in the diglyceride lipase pathway, 1,2-diglyceride and 2-monoglyceride, both accumulated after thrombin stimulation of intact platelets treated with RHC 80267, and arachidonate release was not inhibited. We conclude that RHC 80267 cannot be used to evaluate the diglyceride lipase pathway in intact platelets.     

Hormone-sensitive lipase of adipose tissue.

Some physiologic aspects of the mobilization and fate of free fatty acids are reviewed. The molecular mechanism of the activation of hormone-sensitive lipase in adipose tissue is then discussed. Recent evidence established that hormone-sensitive lipase, concerned with fat mobilization, is both functionally and immunochemically distinct from lipoprotein lipase, concerned with uptake of plasma triglycerides. Lipoprotein lipase activity is not altered by cyclic AMP-dependent protein kinase. The latter enzyme enhances not only triglyceride hydrolase but also monoglyceride, diglyceride and cholesterol ester hydrolase activities in chicken adipose tissue. Finally, it is shown that the activation of all four acyl hydrolases is reversible, the deactivation being magnesium-dependent. Protein phosphatase fractions from heart and liver active against phosphorylase a can reversibly deactivate adipose tissue hormone-sensitive lipase, implying a low degree of substrate specificity for lipase phosphatase.     

Assessment of Noradrenaline and 5-Hydroxytryptamine Uptake in Intact Rat Brain

A method for the measurement of 3H-monoamine [noradrenaline (NA) and 5-hydroxytryptamine (5-HT)] uptake into intact brain following intracerebroventricular injection is described. Most of the accumulated monoamine was associated with nerve terminals (synaptosomes). Radioactivity in the synaptosomal fraction was retained by 0.45-micron filters and was osmotically sensitive, features indicative of accumulation within particles rather than binding to membranes. [3H]5-HT associated with synaptosomes was reduced in animals pretreated with the neurotoxin 5,7-dihydroxytryptamine, in parallel with a reduction in endogenous 5-HT levels. Oral administration of tricyclic antidepressants including clomipramine, desipramine, and imipramine inhibited the synaptosomal accumulation of 3H-monoamines. Overall, the features of the accumulation of 3H-monoamines, after intracerebroventricular administration, displayed many of the characteristics expected of a physiological monoamine reuptake system.     

An in vitro model to study adipose differentiation in serum-free medium

Adipose differentiation was studied in a teratoma-derived fibroadipogenic cell line (1246) cultured in serum-free medium. The addition of dexamethasone and 1-methyl-3-isobutylxanthine to the serum-free medium induced confluent 1246 cells to differentiate into adipocyte-like cells as evidenced by triglyceride accumulation and increased levels of lipolytic enzyme activities. Hormone-sensitive lipase activity measured 5 days after the addition of dexamethasone and 1-methyl-3-isobutylxanthine increased 17-fold and was activated by cAMP-dependent protein kinase. Neutral diglyceride lipase, monoglyceride lipase, and cholesterol ester hydrolase specific activities increased 23-, 75-, and 73-fold, respectively. Among these three activities, only cholesterol ester hydrolase was activated by cAMP-dependent protein kinase. Differentiated 1246 cells expressed receptors to lipolytic hormones as shown by the stimulation of glycerol release by epinephrine (8.6-fold), glucagon (2.2-fold), and adrenocorticotrophic hormone (5.5-fold). Heparin treatment of 1246 cells in serum-free medium resulted in the release of lipoprotein lipase activity into the culture medium. Thus, 1246 cells can serve as a model for the study of adipose differentiation under defined culture conditions since they are capable of growth and survival in the absence of serum while retaining their ability to differentiate into adipocytes.     

The activities of lipases and carnitine palmitoyl-transferase in muscles from vertebrates and invertebrates

1. The activities of tri-, di- and mono-glyceride lipase and carnitine palmitoyltransferase were measured in homogenates of a variety of muscles. These activities were used to estimate the rate of utilization of glycerides and fatty acids by muscle. In muscles whose estimated rates of fat utilization can be compared with rates calculated for the intact muscle from such information as O₂ uptake, there is reasonable agreement between the estimated and calculated rates. 2. In all muscles investigated the maximum rates of hydrolysis of glycerides increase in the order triglyceride, diglyceride, monoglyceride. The activity of diglyceride lipase is highest in the flight muscles of insects such as the locust, waterbug and some moths and is lowest in the flight muscles of flies, bees and the wasp. These results are consistent with the utilization of diglyceride as a fuel for some insect flight muscles. 3. In many muscles from both vertebrates and invertebrates the activity of glycerol kinase is similar to that of lipase. It is concluded that in these muscles the metabolic role of glycerol kinase is the removal of glycerol produced during lipolysis. However, in some insect flight muscles the activity of glycerol kinase is much greater than that of lipase, which suggests a different role for glycerol kinase in these muscles.     

Activation of chicken adipose tissue diglyceride lipase by cyclic AMP-dependent protein kinase and its deactivation by purified protein phosphatase.

Diglyceride lipase of chicken adipose tissue was found to be activated by cyclic AMP-dependent protein kinase to the same extent as hormone-sensitive triglyceride lipase (3-to 10-fold) when lipase assays were carried out in buffers of low ionic strength. Sodium phosphate (50 mM) or sodium chloride (100 mM) preferentially enhanced the basal (nonactivated) form of diglyceride lipase, which minimized the apparent activation by protein kinase. The activated diglyceride lipase was readily deactivated by a pure protein phosphatase from bovine heart (MW 35,000) and the deactivated enzyme was then reactivated by protein kinase.     

FINE STRUCTURAL LOCALIZATION OF ACYLTRANSFERASES : The Monoglyceride and α-Glycerophosphate Pathways in Intestinal Absorptive Cells

A study of the fine structural localization of the acyltransferases of the monoglyceride and α-glycerophosphate pathways for triglyceride synthesis in the intestinal absorptive cell is reported. Glutaraldehyde-fixed tissue was found to synthesize diglyceride and triglyceride from monopalmitin and palmityl CoA, and parallel morphological studies showed the appearance of lipid droplets in the smooth endoplasmic reticulum of the absorptive cell. Glutaraldehyde-fixed tissue also synthesized triglyceride from α-glycerophosphate, although this enzyme system was more susceptible to fixation than the monoglyceride pathway acyltransferases. Cytochemical methods for the localization of free CoA were based (a) on the formation of the insoluble lanthanium mercaptide of CoA and (b) on the reduction of ferricyanide by CoA to yield ferrocyanide which forms an insoluble precipitate with manganous ions. By these methods the monoglyceride pathway acyltransferases were found to be located mainly on the inner surface of the smooth endoplasmic reticulum. The α-glycerophosphate pathway acyltransferases were localized mainly on the rough endoplasmic reticulum. Activity limited to the outer cisternae of the Golgi membranes occurred with both pathways. The possible organization of triglyceride absorption and chylomicron synthesis is discussed in view of these results.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Obtaining monoglycerides by esterification of glycerol with palmitic acid using some high activity preparations of Candida antarctica lipase B

Cross-linked enzyme aggregates (CLEAs), protein coated microcrystals (PCMCs), cross-linked protein coated microcrystals (CLPCMCs) of Candida antarctica lipase B (CALB) were used for esterification of glycerol with palmitic acid in acetone under low water condition. With CLEAs, 81% monoglyceride (MG) along with 4.5% diglyceride (DG) were produced at 1% (v/v) water content in 24 h. The water content in the medium was managed by stepwise addition of the molecular sieves at appropriate time intervals. With PCMCs (potassium sulfate as a core material), 82% monoglyceride along with 4.0% diglyceride were obtained, with 0.5% water (v/v) added initially to anhydrous acetone with molecular sieves present in the reaction medium. With CLPCMC (prepared by cross-linking with 200 mM glutaraldehyde), 87% monoglyceride and 3.3% diglyceride were produced in 24 h in presence of 1% (v/v) water (added initially) and with appropriate amount of molecular sieves added in the reaction medium. The results offer a comparative study on the performance of three high activity preparations of CALB for preparation of monopalmitin with ≤10% of the diglyceride content.     

Triglyceride metabolism in 3T3-L1 cells. An in vivo 13C NMR study.

13C nuclear magnetic resonance spectroscopy has been used to study triglyceride metabolism in 3T3-L1 cells incubated with [1-13/14C] acetate, myristate, palmitate, stearate, or oleate. Labeled cells embedded in agarose filaments were perfused in a specially fitted NMR tube within the spectrometer magnet. Incubation of 3T3-L1 cells with a specific fatty acid enriched the cellular triglycerides with that fatty acid; the NMR signal observed in the carbonyl region of the cell spectrum was due in large part to that fatty acid. NMR data demonstrated that cellular enzymes preferentially esterified saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position. cellular triglyceride hydrolysis by hormone-sensitive lipase was monitored by measuring the decrease in the integrated intensities of resonances arising from fatty acyl carbonyls esterified at glycerol carbons sn-1,3 and sn-2. Under basal conditions, the time courses were first-order, and the average rates were 0.14% of signal/min at both carbonyl positions. Under isoproterenol stimulated conditions, these rates were still first-order and increased 6.4-fold at the sn-1,3 position and 2.4-fold at the sn-2 position. The observation that the hydrolysis time courses were first-order suggested that only a small amount of cellular triglyceride was available to hormone-sensitive lipase, supporting the view that lipolytic enzymes operate at lipid surfaces where only small amounts of neutral lipid may be soluble. Attempts to correlate the measured rates with the rates of hydrolysis at the sn-1,3 and sn-2 positions were hindered by the fact that the chemical shifts of the carbonyl carbons of the diglyceride hydrolysis product did not overlie those of the triglyceride. Analysis of hydrolysis kinetics revealed that hormone-sensitive lipase exhibited little preference for a particular esterified fatty acid under basal conditions; however, under stimulated conditions, the enzyme exhibited a preference for certain triglyceride species.