Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ.

Fumonisins, mycotoxins produced by Fusarium moniliforme and a number of other fungi, cause neuronal degeneration, liver and renal toxicity, cancer, and other injury to animals. Recent work with rat hepatocytes (Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991) J. Biol. Chem. 266, 14486-14490) found that fumonisins block sphingosine biosynthesis by inhibiting the conversion of sphinganine to dihydroceramides, which precedes introduction of the 4,5-trans-double bond of sphingosine. The current study utilized mouse cerebellar neurons in culture to evaluate how this affects the distribution of newly synthesized ceramides among different complex sphingolipids. Fumonisin B1 inhibited ceramide synthase in mouse brain microsomes with a competitive-like kinetic behavior with respect to both sphinganine and stearoyl-CoA. Fumonisin B1 inhibited sphingolipid biosynthesis in cultured cerebellar neurons in situ as reflected by accumulation of free sphinganine, a reduction in the mass of total sphingolipids, reductions in the incorporation of [14C]serine into glucosylceramide, lactosylceramide, sphingomyelin, and gangliosides (GM1, GD3, GD1a, GD1b, GT1b, and GQ1b), and inhibition of the incorporation of [14C]galactose and [3H]sphinganine into complex sphingolipids. Dose-response studies revealed that the labeling of sphingomyelin (IC50 of 0.7 microM) was more sensitive to inhibition by fumonisin B1 than was glycolipid formation (IC50 of approximately 7 microM) in these cells. A similar effect was seen when beta-fluoroalanine was added to inhibit the activity of serine palmitoyltransferase, the first enzyme of the pathway. The inhibition of complex sphingolipid synthesis was reversible, and nearly normal labeling profiles were obtained 48 h after removing the mycotoxin. These studies establish that fumonisin B1 inhibits de novo sphingolipid biosynthesis by neuronal cells and, moreover, that limiting ceramide synthesis differentially affects the formation of sphingomyelin versus glycosphingolipids.

Over-expression of a functionally active human GM2-activator protein in Escherichia coli.

The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator.

Biosynthesis of sphingolipids: dihydroceramide and not sphinganine is desaturated by cultured cells.

Radioactively labeled N-[1-14C]-octanoyl-sphinganine and D-erythro-[3-3H]-sphinganine were administered in parallel experiments to neuroblastoma cells B 104. A time dependent formation of ceramide with a double bond in its sphingoid backbone was observed in both cases. In the presence of fumonisin B1 (25 microM), a strong inhibitor of sphinganine N-acyltransferase, desaturated ceramide was formed only when cells were fed with N-[1-14C]-octanoyl-sphinganine but not with [3-3H]-sphinganine. Thus, the introduction of the double bond occurs only at the level of dihydroceramide, after N-acylation of sphinganine. It is now obvious that sphingosine is not a biosynthetic intermediate but exclusively a catabolic product of cellular sphingolipids.

Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.

Fractionation of primary cultured cerebellar neurons: distribution of sialyltransferases involved in ganglioside biosynthesis.

Primary cultured neurons were fractionated using sucrose density gradients. The activities of four sialyltransferases (GM3, GD3, GD1a, and GT1a synthase) involved in ganglioside biosynthesis were assayed in the collected fractions. The distribution of GM3 synthase coincided with that of mannosidase II, an enzyme assumed to be a cis-Golgi marker. Both enzymes were mainly associated with the more dense fraction. GD1a and GT1a synthase activities, on the other hand, were mainly recovered in the less dense fraction. Moreover, they were colocalized with thiamine pyrophosphatase, an enzyme assumed to be a marker of the late Golgi (trans-Golgi and trans-Golgi network). GD3 synthase activity was equally distributed between both fractions. These results are integrated in a model of ganglioside biosynthesis.

Sphingolipid biosynthesis in cultured neurons. Down-regulation of serine palmitoyltransferase by sphingoid bases.

Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 microM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 microM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 microM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.

Substrate specificity of alpha 2----3-sialyltransferases in ganglioside biosynthesis of rat liver golgi.

The acceptor specificities of four sialytransferases (I, II, IV and V) involved in ganglioside biosynthesis were studied in Golgi vesicles derived from rat liver. The activities of these sialytransferases were strongly detergent-dependent. Competition experiments with different detergent concentrations using LacCer (Gal beta 1----4Glc beta 1----1Cer), GM1a [Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer] and GD1b [Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer] as substrates, and as mutual inhibitors for ganglioside sialyltransferase activity, suggested that sialyltransferase IV was able to catalyze the sialyltransfer in alpha 2----3 linkage to the galactose residues of LacCer as well as of GM1a and GD1b. The other three sialyltransferases (I, II and V) seemed to be quite specific for their respective glycolipid acceptors, LacCer, GM3 and GM1b, GD1a and GT1b. Furthermore the kinetic data showed that sialyltransferase I was inactive at higher detergent concentrations (greater than 75 micrograms Triton CF-54); under these conditions, formation of GM3 and GD1a was catalyzed only by sialyltransferase IV. These results have been integrated into a model for ganglioside biosynthesis and its regulation.

pH-dependent changes of ganglioside biosynthesis in neuronal cell culture.

Ganglioside biosynthesis was studied in primary cultured murine cerebellar cells after labeling with [14C]galactose. A shift in biosynthesis from "a"-series to "b"-series gangliosides was observed after lowering the pH of the culture medium from 7.4 to 6.2; this effect was fully reversible on changing back to pH 7.4. The observed regulatory effect of pH is in accordance with a recent model of ganglioside biosynthesis. Sialyltransferase II (ST II), the first enzyme for biosynthesis of "b"-series gangliosides, is more active at pH 6.2 than Gal-NAc-transferase, the first enzyme for synthesis of "a"-series gangliosides, which is more active than sialyltransferase II at pH 7.4.

Modulation of sphingolipid biosynthesis in primary cultured neurons by long chain bases.

Sphingolipid biosynthesis was studied in cultured murine cerebellar cells in the absence and presence of exogenous sphingosine homologues with different alkyl chain lengths (12, 18, and 24 carbon atoms). Labeling of cells with [14C]serine for 24 h indicated that endogenous sphingosine biosynthesis with incorporation of radiolabeled serine was inhibited by these long chain bases (0.5-50 microM) in a concentration-dependent manner; the inhibition was fully reversible after removal of the long chain bases from the culture medium. Metabolic labeling of neurons with [14C]galactose provided strong evidence that the cells were able to use the exogenous sphingosine homologues, irrespective of their alkyl chain length, as substrates for the biosynthesis of glycosphingolipids. When the biosynthetically inert sphingoid, azidosphingosine (5-50 microM), was fed to the cells, de novo sphingosine and glycosphingolipid biosynthesis were both strongly inhibited.

Uncoupling of ganglioside biosynthesis by Brefeldin A.

We have studied the effect of Brefeldin A (BFA), an antiviral antibiotic, on glycosphingolipid metabolism in primary cultured cerebellar cells. Cells were labeled metabolically with [14C]galactose, or pulse-labeled with precursors of glycosphingolipid biosynthesis; i.e., [14]serine, [3H]palmitic acid or [3H]sphingosine. In all cases BFA (1 microgram/ml) strongly inhibited (75-95%) ganglioside biosynthesis beyond the stage of GM3 and GD3, that is the formation of GM1, GD1a, GT1b and GQ1b. Simultaneously an accumulation of GlcCer, LacCer, GM3 and GD3 was observed (up to 2000%). These effects could be reversed fully by removal of the BFA from the culture medium. These results indicate that the LacCer-, GM3- and GD3-synthases of murine cerebellar cells are localized together on the proximal site of the Golgi apparatus, probably in the cis-Golgi compartment. It is probable that sphingomyelin synthase and some of the other glycosyltransferases involved in ganglioside biosynthesis are localized in distinct compartments beyond the cis Golgi.

Modulation of ganglioside biosynthesis in primary cultured neurons.

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.

Down-regulation of insulin receptors in the heart: studies on primary cultured adult cardiac myocytes.

Primary cultured cardiac myocytes from adult rats have been used to study insulin receptor regulation. Culturing of cells in the presence of insulin induced a dose-dependent down-regulation of insulin binding with a maximal effect of 35% at an insulin concentration of 1.7 X 10(-7) mol/l. The number of high-affinity sites decreased from 110 000 to 70 000 sites per cell in control and down-regulated cells, respectively, with no change in the apparent affinity constant. Down-regulation was found to be rapid (t 1/2 = 3 h) and fully reversible. Culturing of cells in the presence of cycloheximide (0.1 mmol/l) or Tris (35 mmol/l) resulted in a further time-dependent increase in insulin-induced receptor loss with no effect on insulin binding to control cells. The action of both agents was found to be additive reaching a down-regulation of 51% after a culture period of 16 h. Recovery of insulin binding activity after removal of insulin remained unaffected in the presence of cycloheximide, whereas Tris inhibited this process by 74%. In conclusion our results show that the concept of insulin-induced receptor down regulation can be extended to the adult heart muscle. Moreover the data suggest involvement of protein synthesis and receptor recycling in this process.

Adult cardiac myocytes in primary culture: cell characteristics and insulin-receptor interaction.

Calcium-tolerant adult cardiac myocytes were kept in culture under serum-free conditions in the presence of physiological concentrations of insulin. Up to 4 days, 70% of cells retained their in vivo rodshaped morphology without gross structural alterations. During that period a constant ATP-to-ADP ratio was observed with a mean value of 10.6 +/- 0.5 (n = 4). The rate of [14C]phenylalanine incorporation remained unaltered up to 63 h in culture. Insulin binding to cultured cells was found to be time-and temperature-dependent, reversible, and highly specific. Scatchard analysis of equilibrium binding data showed a curvilinear plot with a high-affinity segment yielding an apparent dissociation constant of 4.5 X 10(-10) mol/l and a receptor number of 125,000 sites/cell. Both affinity and receptor number remained unaltered between 18 and 66 h in culture. [14C]phenylalanine incorporation was stimulated by 108% in cardiocytes cultured in the presence of high concentrations of insulin (1.7 X 10(-7) mol/l) for 63 h, when compared with control cells cultured in the absence of insulin. These data demonstrate the retention of structural integrity, insulin receptors, and insulin responsiveness in primary cultured adult cardiac myocytes and provide a useful model for long-term studies on the regulation of insulin action on the heart.