SMS [SM (sphingomyelin) synthase] is a class of enzymes that produces SM by transferring a phosphocholine moiety on to ceramide. PC (phosphatidylcholine) is believed to be the phosphocholine donor of the reaction with consequent production of DAG (diacylglycerol), an important bioactive lipid. In the present study, by modulating SMS1 and SMS2 expression, the role of these enzymes on the elusive regulation of DAG was investigated. Because we found that modulation of SMS1 or SMS2 did not affect total levels of endogenous DAG in resting cells, whereas they produce DAG in vitro, the possibility that SMSs could modulate subcellular pools of DAG, once acute activation of the enzymes is triggered, was investigated. Stimulation of SM synthesis was induced by either treatment with short-chain ceramide analogues or by increasing endogenous ceramide at the plasma membrane, and a fluorescently labelled conventional C1 domain [from PKC (protein kinase C)] enhanced in its DAG binding activity was used to probe subcellular pools of DAG in the cell. With this approach, we found, using confocal microscopy and subcellular fractionation, that modulation of SMS1 and, to a lesser extent, SMS2 affected the formation of DAG at the Golgi apparatus. Similarly, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein PKD (protein kinase D) to the Golgi. These results provide direct evidence that both enzymes are capable of regulating the formation of DAG in cells, that this pool of DAG is biologically active, and for the first time directly implicate SMS1 and SMS2 as regulators of DAG-binding proteins in the Golgi apparatus.

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Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.

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The purpose of this study was to examine the effects of DL: -alpha-lipoic acid (LA) on arsenic (As) induced alteration of glutathione (GSH) level and of the activity of glutathione-related enzymes-glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH)-in rat brain regions (cortex, hypothalamus, striatum, cerebellum and hippocampus). Male Wistar rats of 150+/-10 g weight were divided into four groups: control and three experimental groups supplemented with arsenic (sodium arsenite) alone (100 ppm mixed in drinking water), lipoic acid alone (70 mg kg(-1) body weight), arsenic plus lipoic acid (100 ppm arsenic in drinking water plus 70 mg lipoic acid kg(-1) body weight). The arsenic content of brain regions was found to increase with the administration of sodium arsenite. Arsenic exposure elicited a significant decline in glutathione content and in the activity of related enzymes, with the greatest decreases seen in the cortex, striatum, and hippocampus, whereas there were no significant differences between control rats and the group treated with lipoic acid alone. Highly elevated content of the thiobarbituric acid-reactive substance malondialdehyde (MDA) in the brain regions of arsenic-exposed rats reflected extensive lipid peroxidation (LPO) processes. Simultaneous lipoic acid treatment was effective in reducing brain regional arsenic levels and lipid peroxidation and in increasing the glutathione content and the activity of its related enzymes. Lipoic acid, by acting as an alternative sulfhydryl nucleophile to glutathione, prevents its oxidation to glutathione disulfide in detoxifying reactions against reactive oxygen species and consequently increases the activity of glutathione-related enzymes.