Formation of phosphatidylethanol in rat brain by phospholipase D

The mechanism of phosphatidyl [14C]ethanol formation was studied in rat brain microsomal fraction. Phospholipase D and base-exchange enzymes were assayed with [14C]ethanol as substrate. Phospholipase D was found to catalyse the formation of phosphatidylethanol. The reaction was dependent on sodium-oleate as activating factor. Phosphatidylethanol formation by phospholipase D has previously only been reported to occur in plant tissues. Stimulation of base-exchange enzymes with calcium in the presence of [14 C]ethanol did not induce any formation of phosphatidylethanol. These findings indicate that phosphatidylethanol formation in ethanol intoxicated rats is catalysed by phospholipase D.     

Production of the glycosylphosphatidylinositol-specific phospholipase D by the islets of Langerhans.

A large number of diverse cell surface proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. One proposed function for the GPI anchor is that it facilitates the release of the protein from the cell by acting as a target for anchor-specific phospholipases. We and others have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD) (Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem, Biophys. Res. Commun. 150, 476-482; Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984). Because the GPI-PLD recognizes a conserved portion of the anchor, all GPI-anchored proteins are potential substrates for the enzyme. We demonstrate in this communication the production of the plasma GPI-PLD by the islets of Langerhans. GPI-PLD enzymatic activity was found in dog pancreatic microsomes, but not pancreatic juice. Both the pancreatic and plasma enzymes were divalent cation-dependent and had identical substrate specificities. Purified murine islets of Langerhans, as well as alpha and beta cells, contained and released GPI-PLD activity. A GPI-PLD DNA fragment was amplified by polymerase chain reaction from a normal human islet cDNA library; the amplified fragment hybridized with the GPI-PLD cDNA clone. These findings represent the first demonstration of the production of the plasma GPI-PLD by a specific tissue site as well as cell type.     

Stimulation by glucose and carbamylcholine of phospholipase C in pancreatic islets

Phosphoinositide hydrolysis in intact pancreatic islet cells was investigated in an indirect but dynamic manner by monitoring the efflux of radioactivity from islets prelabelled with [3H]inositol. A rise in glucose concentration provoked a rapid, modest but sustained increase in effluent radioactivity, this phenomenon being abolished in the absence of extracellular Ca2+ or presence of verapamil. The release of [3H]inositol was also stimulated at high extracellular K+ concentration, but not by gliclazide. Whether in the presence or absence of glucose, carbamylcholine provoked a marked increase in effluent radioactivity. The response to the cholinergic agent was decreased in the presence of verapamil or absence of extracellular Ca2+ and abolished in the presence of atropine or LiCl. These results suggest that an increase in cytosolic Ca activity, as caused by glucose or membrane depolarization, may cause activation of phospholipase C. In response to cholinergic agents, however, the enzymic activation, although modulated by Ca2+ availability, may result directly from the occupation of muscarinic receptors.     

Phosphatidylethanol biosynthesis in ethanol-exposed NG108-15 neuroblastoma X glioma hybrid cells. Evidence for activation of a phospholipase D phosphatidyl transferase activity by protein kinase C

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the release of free choline from intact NG108-15 cells into the medium, without affecting the release of phosphocholine (Liscovitch, M., Blusztajn, J.K., Freese, A., and Wurtman, R.J. (1987) Biochem. J. 241, 81-86). To test the hypothesis that this response reflects activation of cellular phospholipase D, via protein kinase C (Ca2+/phospholipid-dependent enzyme), I examined in NG108-15 cells the biosynthesis of the abnormal phospholipid phosphatidylethanol, produced by phospholipase D in the presence of ethanol by transphosphatidylation. Phosphatidylethanol production was quantitated by measuring the incorporation of phosphatidyl moieties (prelabeled metabolically with [3H]oleic acid) into phosphatidylethanol. The production of phosphatidylethanol in NG108-15 cells was virtually dependent on stimulation by TPA, in a time- and concentration-dependent manner (EC50 = 18 nM). The rate of 3H-phosphatidylethanol formation reached a peak after 10 min of incubation with TPA and declined gradually thereafter. The levels of 3H-phosphatidylethanol in TPA-treated cells were directly related to ethanol concentration in the physiologically attainable range (20-80 mM). Phosphatidylethanol production was activated only by phorbol derivatives that are activators of protein kinase C (i.e. TPA, 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate) and could be mimicked by a cell-permeant diacylglycerol, 1,2-dioctanoyl-sn-glycerol, in a nonadditive manner. The effect of TPA was inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (0.1 mM) by 70% but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide. Phosphatidylethanol formation was completely abolished in cells in which protein kinase C was down-regulated by pretreatment of the cells with TPA. These results indicate that phosphatidylethanol biosynthesis in NG108-15 cells depends largely on activation of protein kinase C. In contrast to its effects on the release of free choline and on the accumulation of phosphatidylethanol, TPA did not affect the levels of phosphatidic acid in NG108-15 cells. It is therefore proposed that protein kinase C selectively activates the phosphatidyl transferase activity of phospholipase D, reflecting a signal termination mechanism which may be operative in phospholipase D-mediated signal transduction cascades.     

Hexose metabolism in pancreatic islets: regulation of mitochondrial hexokinase binding

A major fraction of hexokinase was found to be bound, presumably to mitochondria, in both normal and tumoral rat pancreatic islet cells examined after either mechanical disruption or digitonin treatment. Spermidine enhanced the binding and glucose 6-phosphate caused the release of hexokinase to and from islet mitochondria, in a manner comparable to that seen in parotid or brain homogenates. In hepatocytes, some hexokinase, but no glucokinase, was found in the bound form. In islet cells, however, the pattern of glucokinase binding was similar to that of hexokinase. It is speculated that the preferential location of both hexokinase and glucokinase on mitochondria may favor the maintenance of a high cytosolic ATP content in islet cells.     

Encapsulation of pancreatic islets for transplantation in diabetes: the untouchable islets

The aim of encapsulation of pancreatic islets is to transplant in the absence of immunosuppression. It is based on the principle that transplanted tissue is protected from the host immune system by an artificial membrane. Encapsulation allows for application of insulin-secreting cells of animal or other surrogate sources, to overcome human islet shortage. The advantages and pitfalls of the approaches developed so far are discussed and compared, together with some recent progress, in view of applicability in clinical islet transplantation.     

The formation of phosphatidylglycerol and other phospholipids by the transferase activity of phospholipase D

1. Purified phospholipase D can catalyse the transfer of a `phosphatidyl' unit from lecithin to various aliphatic alcohols such as glycerol, ethanolamine, methanol and ethylene glycol with the formation of the equivalent phospholipid. 2. The transferase reaction occurs simultaneously with hydrolase activity but at high alcohol concentrations the former predominates. 3. The acceptor molecule must contain a primary alcoholic grouping. 4. The chromatographic and ionophoretic mobilities of the deacylation products of many enzymically synthesized phospholipids are reported. 5. Enzymically prepared phosphatidylglycerol has been isolated in good yield. Chemical degradation showed that the `phosphatidyl unit' of lecithin had been transferred predominantly to the α-hydroxyl groups of glycerol. 6. Water-soluble alcohols can markedly stimulate the liberation of choline from ultrasonically treated lecithin by phospholipase D. The stimulation is usually due to an increase in hydrolase activity although often the associated transferase activity contributes.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Hexose metabolism in pancreatic islets: compartmentation of hexokinase in islet cells

Hexokinase activity was found in both soluble (cytosolic) and particulate subcellular fractions prepared from rat pancreatic islet homogenates. The bound enzyme was associated with mitochondria rather than secretory granules. Relative to the total hexokinase activity, the amount of bound enzyme was higher in islet homogenates prepared at pH 6.0 (72 +/- 7%) than in islets homogenized at pH 7.4 (38 +/- 1%). The affinity of hexokinase for equilibrated D-glucose was not different in the cytosolic and mitochondrial fractions. In both fractions, hexokinase displayed a greater affinity for alpha- than beta-D-glucose, but a higher maximal velocity with the beta- than alpha-anomer. Glucose 6-phosphate inhibited to a greater extent cytosolic than mitochondrial hexokinase. A high Km glucokinase-like enzymic activity was also present in both subcellular fractions. It is proposed that the ambiguity of hexokinase plays a propitious role in the glucose-sensing function of pancreatic islet cells.     

Prostaglandin synthesis and metabolism in isolated pancreatic islets of the rat

Isolated pancreatic islets of Langerhans of the rat which were sonicated and incubated with radiolabeled arachidonic acid for 1 hr synthesized several species of prostaglandins (PGs). Both thin-layer and high-performance liquid (HPLC) chromatographic techniques demonstrated the synthesis by islet sonicates of PGF_2α and PGE₂ equivalents, in addition to the 15-keto-13, 14-dihydro metabolites of these primary PGs. In addition, HPLC allowed the identification of 6-keto-PGF_1α (the metabolite of prostacyclin) as a major PG synthesized from arachidonate by this tissue. Islet vascular elements, as well as endocrine cells, may contribute to the synthesis of the latter compound. Lesser amounts of arachidonate were incorporated into PG-like compounds eluting as thromboxane. The synthesis of PGs was sensitive to the protein concentration of islet sonicate, and a five-fold dilution of protein resulted in a comparable reduction in arachidonate incorporation into PGs. Labeled arachidonate was also incorporated into compounds which elute as hydroxy or hydroperoxy-eicosatetrainoic acids on HPLC. Thus, isolated pancreatic islets synthesize a variety of PGs which may have a physiological role in hormone secretion form this endocrine organ.     

Hexose metabolism in pancreatic islets: Effect of D-glucose on the mitochondrial redox state

The mitochondrial NADH/NAD⁺ ratio for free nucleotides in rat pancreatic islets was judged from the cell content in L-glutamate and L-alanine, 2-ketoglutarate and pyruvate, and NH ₄ ⁺ . At a physiological concentration of D-glucose, such a ratio averaged 9.6±1.1%. A rise in hexose concentrations, above a threshold value in excess of 5.6 mM, caused a rapid, sustained and rapidly reversible decrease in the mitochondrial NADH/NAD⁺ ratio. It is speculated that in the process of glucose-stimulated insulin release, the latter change participates in the coupling between metabolic and secretory events by favouring both the activity of key mitochondrial dehydrogenases and the translocation of Ca²⁺ from the mitochondria into the cytosol.     

A role for calcium in the breakdown of inositol phospholipids in intact and digitonin-permeabilized pancreatic islets.

Glucose (20 mM) and 4-methyl-2-oxopentanoate (10 mM) both caused a pronounced stimulation of insulin release and of [3H]inositol phosphate production in rat pancreatic islets prelabelled with myo-[3H]inositol. Secretory responses to these nutrients were markedly impaired by lowering the Ca2+ concentration of the incubation medium to 10(-4)M or less, whereas stimulated inositol phosphate production was sensitive to Ca2+ within the range 10(-6)-10(-4)M. Inositol phosphate formation in response to carbamoylcholine was also found to be dependent on the presence of 10(-5)M-Ca2+ or above. Raising the concentration of K+ in the medium resulted in a progressive, Ca2+-dependent stimulation of inositol phosphate production in islets, although no significant stimulation of insulin release was observed. In islets prelabelled with myo[3H]inositol, then permeabilized by exposure to digitonin, [3H]inositol phosphate production could be triggered by raising the Ca2+ concentration from 10(-7) to 10(-5)M. This effect was dependent on the concentration of ATP and the presence of Li+, and involved detectable increases in the levels of InsP3 and InsP2 as well as InsP. A potentiation of inositol phosphate production by carbamoylcholine was observed in permeabilized islets at lower Ca2+ concentrations, although nutrient stimuli were ineffective. No significant effects were observed with guanine nucleotides or with neomycin, although NADH produced a modest increase and adriamycin a small inhibition of inositol phosphate production in permeabilized islets. These results strongly suggest that Ca2+ ions play an important role in the stimulation of inositol lipid metabolism in islets in response to nutrient secretagogues, and that inositide breakdown may actually be triggered by Ca2+ entry into the islet cells.     

Hexose metabolism in pancreatic islets: enzyme-to-enzyme tunnelling of hexose 6-phosphates.

The fate of unlabelled D-glucose and D-[2-3H]glucose in pancreatic islets was simulated taking into account experimental values for glycolytic flux, intracellular concentration of D-glucose 6-phosphate and phosphoglucoisomerase activity. The model, which also takes into account the isotopic discrimination in velocity and intramolecular transfer of tritium between D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase, revealed that the predicted generation of 3HOH from D-[2-3H]glucose was much higher than the true experimental value. Such a discrepancy is reinforced by the consideration that the generation of 3HOH from D-[2-3H]glucose in islet cells is not solely attributable to the phosphoglucoisomerase-catalyzed detritiation of hexose 6-phosphates metabolized in the glycolytic pathway. In order to reconcile experimental and theoretical values for 3HOH production, it was found necessary to postulate enzyme-to-enzyme tunnelling of hexose 6-phosphates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. It is proposed that such a tunnelling may favour the anomeric specificity of D-glucose metabolism in islet cells, by restricting the anomerization of hexose 6-phosphates.     

Hexose metabolism in pancreatic islets. Feedback control of D-glucose oxidation by functional events

A rise in extracellular D-glucose concentration in pancreatic islet cells causes a greater relative increase in the oxidation of pyruvate and acetyl residues than in glycolysis. A possible explanation for such an unusual situation was sought in the present study. The preferential stimulation of mitochondrial oxidative events was found to display a sigmoidal dependency on hexose concentration, and an exponential time course during prolonged exposure of the islets to a high concentration of D-glucose. The preferential stimulation of mitochondrial oxidative events was abolished in islets incubated in the presence of cycloheximide and absence of Ca2+, in which case the oxidation of D-[6-14C]glucose was more severely inhibited than that of D-[3,4-14C]glucose. Likewise, the inhibitor of protein biosynthesis and the absence of Ca2+ affected the oxidation of L-[U-14C]leucine preferentially, relative to that of L-[1-14C]leucine, in islets exposed to a high, but not a low, concentration of the amino acid. These results demonstrate that in pancreatic islets it is possible to dissociate both glycolysis from mitochondrial oxidative events and the oxidation of acetyl residues from their generation rate. Moreover, the experimental data suggest that nutrient-responsive and ATP-requiring functional processes exert a feedback control on mitochondrial respiration in this fuel-sensor organ.