Lipolytic action of cholera toxin on fat cells. Re-examination of the concept implicating GM1 ganglioside as the native membrane receptor. (1/175)

The possible role of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1) ganglioside in the lipolytic activity of cholera toxin on isolated fat cells has been examined. Analyses of the ganglioside content and composition of intact fat cells, their membranous ghosts, and the total particulate fraction of these cells indicate that N-acetylneuraminylgalactosylglucosylceramide (GM3) represents the major ganglioside, with substantial amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2) and smaller amounts of other higher homologues also present. Native GM1 was not detected in any of these preparations. Examination of the relative capacities of various exogenously added radiolabeled sphingolipids to bind to the cells indicated that GM2 and glucosylsphingosine were accumulated by the cells to extents comparable to GM1. Galactosylsphingosine and sulfatide also exhibited significant, although lesser, binding affinities for the cells. The adipocytes appeared to nonspecifically bind exogenously added GM1; saturation of binding sites for GM1 could not be observed up to the highest concentration tested (2 X 10(-4) M), wherein about 7 X 10(9) molecules were associated with the cells. Essentially all of this exogenously added GM1 was found bound to the plasma membrane "ghost" fraction. Investigation of the biological responses of the cells confirmed their sensitivities to both cholera toxin and epinephrine-stimulated lipolysis, as well as the lag period displayed during the toxin's action. While we could confirm that the toxin's lipolytic activity can be enhanced by prior treatment of the fat cells with GM1, several of the observed characteristics of this phenomenon differ from earlier reported findings. Accordingly, added GM1 was able to enhance only the subsequent rate, but not the extent, of toxin-stimulated glycerol release (lipolysis) from the cells. We also were unable to confirm the ability of GM1 to enhance the toxin's activity at either saturating or at low toxin concentrations. The limited ability of added GM1 to enhance the toxin's activity appeared in a unique bell-shaped dose-response manner. The inability of high levels of GM1 to stimulate a dose of toxin that was ineffective on native cells suggests that the earlier reported ability of crude brain gangliosides to accomplish this was due to some component other than GM1 in the crude extract. While several glycosphingolipids and some other carbohydrate-containing substances that were tested lacked the ability to mimic the enhancing effect of GM1, 4-methylumbelliferyl-beta-D-galactoside exhibited an effect similar to, although less pronounced than, that of GM1. The findings in these studies are unable to lend support to the earlier hypothesis that (a) GM1 is cholera toxin's naturally occurring membrane receptor on native fat cells, and (b) the ability of exogenously added GM1 to enhance the toxin's lipolytic activity represents the specific creation of additional natural receptors on adipocytes...  (+info)

Effect of gemfibrozil in vitro on fat-mobilizing lipolysis in human adipose tissue. (2/175)

Fat-mobilizing lipolysis was studied in rat and human adipose tissue during incubation in vitro by following the release of glycerol into the incubation medium. Gemfibrozil as well as clofibrate consistently and readily inhibited basal as well as noradrenaline-stimulated fat-mobilizing lipolysis in rat fat. With human adipose tissue no effect was observed with gemfibrozil and clofibrate on basal lipolysis. This may be due to the comparatively low rate of the nonstimulated fat-mobilizing lipolysis in human tissue incubated in vitro. When lipolysis was stimulated with noradrenaline as well as isoprenaline, however, both gemfibrozil and clofibrate significantly reduced the fat-mobilizing lipolysis. This inhibition of lipolysis was however not observed in all studies. When lipolysis had been stimulated with theophylline, no inhibition of lipolysis was obtained with either compound. The possibility that reduced fat-mobilizing lipolysis in adipose tissue may cause a lowering of plasma triglycerides by reducing the flow of FFA to the liver is discussed in some detail. It is also suggested that inhibition of lipolysis may be accompanied by increased activity of lipoprotein lipase as well as an increase in the FIAT process. However, the pharmacological implication of the above-mentioned findings, particularly for gemfibrozil, must await further studies, as fairly large doses, around 1 mg/ml of incubation medium, were needed to obtain inhibition of fat-mobilizing lipolysis.  (+info)

Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola. (3/175)

Colletotrichum graminicola, a pathogen of sorghum and corn, was investigated prior and during germination as to certain aspects of acid phosphatase activity and lipid mobilization. Ungerminated conidia cytoplasm was filled with lipid deposits, which were mobilized during the germination process. Cytochemical ultrastructural examination showed that conidia vacuoles exhibit acid phosphatase activity, which is suggestive of lytic activity. Lipid bodies, stored in the ungerminated conidia cytoplasm, were internalized by vacuoles in a process analogous to microautophagy and were apparently digested inside them. The lipid bodies disappeared and vacuoles became enlarged in conidial cells during germination. Appressoria also showed acid phosphatase activity in multiple heterogeneous vesicles which were, in most cases, juxtaposed with lipid bodies. These results suggest that the vacuolar system plays an important role during C. graminicola germination and that the initial stages of lipid metabolization are taking place inside the vacuoles.  (+info)

Partial purification and characterization of a triglyceride lipase from pig adipose tissue. (4/175)

A triglyceride lipase was extracted from defatted pig adipose tissue powder with dilute ammonia and purified about 230-fold by a combination of ammonium sulfate fractionation, heparin-Sepharose 4B, DEAE-cellulose, and Sephadex G-150 column chromatographies and isoelectrofocusing electrophoresis. The enzyme was distinguishable in physical and kinetic properties from the two previously defined lipases in adipose tissue, lipoprotein lipase, and hormone-sensitive lipase. The purified enzyme was fully active in the absence of serum lipoprotein and was not stimulated by adenosine 3':5'-monophosphate-dependent protein kinase. In marked contrast to the already defined lipases, the enzyme was strongly inhibited by serum albumin. The enzyme had a molecular weigt of about 43,000, a pI of 5.2, and pH optimum of 7.0. The enzyme hydrolyzed triolein to oleic acid and glycerol, and did not exhibit esterase activity. The apparent Km for triolein was 0.05 mM. Physiological roles of this new species of lipase remained to be explored.  (+info)

Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol. (5/175)

A microsomal triacylglycerol hydrolase (TGH) was recently purified from porcine liver [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. To gain further insight into the function of TGH, we have cloned a cDNA encoding TGH from a rat liver cDNA library and generated McArdle RH7777 rat hepatoma cell lines that stably express the rat TGH. The putative protein derived from the cDNA sequence contains a cleavable signal sequence and a catalytic site serine residue present within a pentapeptide motif (GXSXG) that is conserved in all known lipases. TGH-transfected cells showed a 2-fold increase, compared with control cells, in the rate of depletion of prelabelled triacylglycerol stores. Thus, TGH is capable of hydrolysis of stored triacylglycerol. In contrast, the rate of turnover of labelled phosphatidylcholine was similar in both the vector- and TGH-transfected cells. Studies in TGH-transfected cells demonstrated that utilization of intracellular triacylglycerol pools for secretion was approx. 30% higher than in vector-transfected cells. Whereas phosphatidylcholine secretion was essentially the same in control and TGH-transfected cells, TGH-transfected cells also secreted an approx. 25% greater mass of triacylglycerol into the medium and had increased levels of apolipoprotein B100 in the very-low-density lipoprotein density range compared with control cells. The results suggest that the microsomal TGH actively participates in the mobilization of cytoplasmic triacylglycerol stores, some of which can be used for lipoprotein assembly.  (+info)

Mobilisation of triacylglycerol stores. (6/175)

Triacylglycerol (TAG) is an energy dense substance which is stored by several body tissues, principally adipose tissue and the liver. Utilisation of stored TAG as an energy source requires its mobilisation from these depots and transfer into the blood plasma. The means by which TAG is mobilised differs in adipose tissue and liver although the regulation of lipid metabolism in each of these organs is interdependent and synchronised in an integrated manner. This review deals principally with the mechanism of hepatic TAG mobilisation since this is a rapidly expanding area of research and may have important implications for the regulation of plasma very-low-density lipoprotein metabolism. TAG mobilisation plays an important role in fuel selection in non-hepatic tissues such as cardiac muscle and pancreatic islets and these aspects are also reviewed briefly. Finally, studies of certain rare inherited disorders of neutral lipid storage and mobilisation may provide useful information about the normal enzymology of TAG mobilisation in healthy tissues.  (+info)

Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells. (7/175)

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.  (+info)

Glucogenic and ketogenic capacities of lard, safflower oil, and triumdecanoin in fasting rats. (8/175)

The glucogenic and ketogenic capicities of lard, safflower oil, and triundecanoin were compared. Rats were fed diets containing 30 percent of either lard (a ketogenic fat), triundercanoin (a glucogenic fat), or safflower oil (a fat high in linoleic acid). After 61 days, the rats were fasted for 72 hours. Plasma glucose and ketone body concentrations and carcass fatty acid loss were measured during fasting. The lard-fed animals, which lost mostly saturated even-chain length fatty acids during fasting, did not maintain their prefasting plasma glucose levels and became ketotic. The animals that had been fed triundecanoin (which mobilized considerable odd-chain fatty acid) maintained their prefasting plasma glucose levels and did not become ketotic. The animals fed safflower oil (which mobilized massive amounts of linoleic acid) showed even lower levels of plasma glucose and higher levels of ketone bodies than did the animals fed lard. This failure of safflower oil to avert fasting hypoglycemia suggests that linoleic acid is oxidized in a manner more like the saturated fatty acid of lard than like the glucogenic odd-chain fatty acid (undecanoic).  (+info)