Early fumonisin B1 toxicity in relation to disrupted sphingolipid metabolism in male BALB/c mice

Fumonisin B₁ is a mycotoxin produced by Fusarium moniliforme, a common fungus in corn. It is known to cause a variety of diseases, including hepatic and renal degeneration in many species of laboratory and domestic animals. The known biochemical events in fumonisin B₁ toxicity involve inhibition of ceramide synthase leading to disruption of sphingolipid metabolism. The effect of fumonisin B₁ on ceramide and more complex sphingolipids in mice is not known. Groups of five male BALB/c mice each were injected with fumonisin B₁ subcutaneously at doses of 0, 0.25, 0.75, 2.25, and 6.75 mg/kg body weight daily for 5 days. This protocol has been shown to produce a dose-dependent increase in apoptosis in liver and kidney of these animals. In the present study, liver, kidney, and brain were sampled and analyzed for free sphingoid bases and complex sphingolipids one day after the last treatment. A dose-related accumulation of free sphinganine and sphingosine was observed in liver and kidney, but not brain. The maximal increase in free sphinganine in kidney was 10-fold greater than in liver. Total phospholipids increased only in liver, whereas ceramide levels were not consistently altered in liver, kidney, or brain. In liver and kidney, fumonisin B₁ treatment increased the sphinganine-containing complex sphingolipids, but no effect was observed on sphingosine-containing complex sphingolipids. No changes in complex sphingolipids were observed in brain. In liver, there was a close correlation between the extent of free sphinganine accumulation, and apoptosis and hepatopathy. This correlation was also evident in kidney but to a lessor extent. Nonetheless, the apoptosis and nephropathy occurred with little or no change in the levels of ceramide or more complex sphingolipids. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 281–289, 1998     

Endogenous levels of 1-O-acylceramides increase upon acidic ceramidase deficiency and decrease due to loss of Dgat1 in a tissue-dependent manner

Except for epidermis and liver, little is known about endogenous expression of 1-O-acylceramides (1-OACs) in mammalian tissue. Therefore, we screened several organs (brain, lung, liver, spleen, lymph nodes, heart, kidney, thymus, small intestine, and colon) from mice for the presence of 1-OACs by LC-MS². In most organs, low levels of about 0.25–1.3 pmol 1-OACs/mg wet weight were recorded. Higher levels were detected in liver, small and large intestines, with about 4–13 pmol 1-OACs/mg wet weight. 1-OACs were esterified mainly with palmitic, stearic, or oleic acids. Esterification with saturated very long-chain fatty acids, as in epidermis, was not observed. Western-type diet induced 3-fold increased 1-OAC levels in mice livers while ceramides were unaltered. In a mouse model of Farber disease with a decrease of acid ceramidase activity, we observed a strong, up to 50-fold increase of 1-OACs in lung, thymus, and spleen. In contrast, 1-OAC levels were reduced 0.54-fold in liver. Only in lung 1-OAC levels correlated to changes in ceramide levels - indicating tissue-specific mechanisms of regulation. Glucosylceramide synthase deficiency in liver did not cause changes in 1-OAC or ceramide levels, whereas increased ceramide levels in glucosylceramide synthase-deficient small intestine caused an increase in 1-OAC levels. Deficiency of Dgat1 in mice resulted in a reduction of 1-OACs to 30% in colon, but not in small intestine and liver, going along with constant free ceramides levels. From these data, we conclude that Dgat1 as well as lysosomal lipid metabolism contribute in vivo to homeostatic 1-OAC levels in an organ-specific manner.     

Enzymic effects of beta-glucosidase destruction in mice. Changes in glucuronidase levels.

1. The injection into mice of a single dose of conduritol B epoxide, a covalent inhibitor of glucosidases, quickly produced changes in tissue levels of beta-D-glucuronidase (EC 3.2.1.31). The specific activity of the enzyme decreased in liver, spleen and kidney while brain showed little change. The inhibitor did not act on glucuronidase in vitro, so the effect of the inhibitor is complex, possibly a result of the loss of glucosidase activity. Since glucuronidase contains glucose, we suggest that the transport of the enzyme between subcellular regions and tissues involves loss of part of the glucose moieties. 2. Levels of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) dropped very rapidly after epoxide injection, reaching a minimum at 1 h in liver. There was a noticeable restoration of activity within the next 1--2 h. Aryl beta-glucosidase (EC 3.2.1.21) decrease somewhat less than cerebrosidase, reaching a minimum within 2 h. It too showed some recovery of activity within 3 h. 3. Acid phosphatase rose slightly in liver but not in brain. alpha-L-Fucosidase and angiotensin-converting enzyme were not affected by the epoxide injection. The latter two enzymes are known to contain glucose. 4. Injection of a hemolyzing agent, phenylhydrazine, produced an increased level of glucuronidase in liver and spleen within 6 days, but not in kidney. This enhancement was a little less in mice previously injected with the glucosidase inhibitor. 5. Mice injected with the epoxide once a day eight times showed a distinct rise in brain glucuronidase level, as well as a rise in brain weight. However, the other organs showed only the same decrease in glucuronidase specific activity noted with the single injection protocol. It is suggested that the difference is due to the blood-brain barrier, which could slow the loss of brain glucuronidase from the extracellular fluid.     

Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.     

Ceramide-like synthetic amides that inhibit cerebroside galactosidase

Amides resembling ceramide (fatty acyl sphingosine) were synthesized and tested for their effects on rat brain cerebrosidase (galactosyl ceramide beta-galactosidase). The best inhibitor was N-decanoyl dl-erythro-3-phenyl-2-aminopro-panediol, which exhibited a K(i) of 0.4 mm. A Lineweaver-Burk plot indicated that the amide acted as a noncompetitive inhibitor, presumably by attachment to a site other than the substrate-active site. Preincubation did not affect the degree of inhibition, and inhibition was independent of incubation duration; these observations suggest that the inhibitor does not combine with the enzyme irreversibly. Structural variations produced decreased inhibitory activity: loss of one of the hydroxyl groups, replacement of the aromatic side chain with an aliphatic or substituted phenyl group, or isomeric inversion of the 3-hydroxyl group. It appears that the best activity is obtained with a substance most closely resembling natural ceramide. The cerebrosidases of rat spleen, kidney, and liver are also inhibited by the same amide.     

Gangliosides Inhibit Glucosylceramide Synthase: A Possible Role in Ganglioside Therapy

Gangliosides stimulate the hydrolysis of glucosylceramide (GlcCer), their precursor, and therefore may lower the level of cellular GlcCer and exert a feedback control effect to slow the formation of gangliosides. Tests were made to see if a similar effect on GlcCer levels can be exerted by the action of gangliosides on GlcCer synthesis. Using a new assay procedure, we showed that gangliosides do inhibit the synthase in brain membranes quite effectively, the most active being those lipids with more sugar and sialic acid moieties. Mice injected with a mixture of brain gangliosides for 5 days were found to have a lower level of ceramide:UDP-Glc glucosyltransferase activity in brain, liver, and kidney. The inhibition seems to be exerted by competition for the active site and binding to effector site(s) on the enzyme. It is possible that the reported therapeutic actions of gangliosides on the nervous system are, in part, the result of lowered levels of GlcCer. Malignant tumors shed gangliosides into the extracellular fluid, which are believed to block the generation of antibodies by the host's immunodefense system; this effect also may be due, in part, to reduction in the GlcCer level of immunogenic cells. A new finding is that a ceramide containing phytosphingosine is a markedly better substrate for GlcCer synthase than one containing the more common base.     

Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells

Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as H(2)O(2), C(6)-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitor dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-d-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by C(6)-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2.     

Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands

The topology of ceramide glucosyltransferase and de novo synthesized glucosylceramide was studied in sealed and 'right-side-out' vesicles of porcine submaxillary glands derived from Golgi apparatus. Pronase treatment which did not cause any breakdown of the luminal glycoprotein galactosyltransferase activity, inhibited the ceramide glucosyltransferase to more than 50% at a ratio proteinase to Golgi protein 1:100. Trypsin at the same concentration, while producing no inactivation of luminal galactosyltransferase, caused a complete loss of ceramide glucosyltransferase activity. The membrane-impermeable compound, DIDS, which did not cause any inhibition of the galactosyltransferase, inhibited the ceramide glucosyltransferase (70% reduction at 80 microM DIDS). Thus, the enzyme ceramide glucosyltransferase is accessible from the cytoplasmic side of the Golgi vesicles. The orientation of the newly synthesized glucosylceramide is studied by the ability of the enzyme glucosylceramidase to hydrolyse this compound both on intact and on disrupted vesicles. The same percentage (respectively, 36 and 30%) of hydrolysis was obtained during an incubation of 3 h, showing that glucosylceramide is not at all protected from external hydrolysis. Pronase-treated vesicles revealed an increase in glucosylceramidase hydrolysis (up to 45%), which indicates that glucosylceramide that glucosylceramide may be cryptic. All these results indicate that the ceramide glucosyltransferase, as well as related glucosylceramide, are cytoplasmically oriented in Golgi vesicles from porcine submaxillary glands.     

Treatment of neutral glycosphingolipid lysosomal storage diseases via inhibition of the ABC drug transporter, MDR1. Cyclosporin A can lower serum and liver globotriaosyl ceramide levels in the Fabry mouse model

We have shown that the ABC transporter, multiple drug resistance protein 1 (MDR1, P-glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, glycosphingolipid (GSL) synthesis. Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumulated glucosyl ceramide and Fabry cell lines of globotriaosyl ceramide (Gb3), by preventing de novo synthesis. In the Fabry mouse model, Gb3 is increased in the heart, liver, spleen, brain and kidney. The lack of renal glomerular Gb3 is retained, but the number of verotoxin 1 (VT1)-staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice. Adult Fabry mice were treated with alpha-galactosidase (enzyme-replacement therapy, ERT) to eliminate serum Gb3 and lower Gb3 levels in some tissues. Serum Gb3 was monitored using a VT1 ELISA during a post-ERT recovery phase +/- biweekly intra peritoneal CsA. After 9 weeks, tissue Gb3 content and localization were determined using VT1/TLC overlay and histochemistry. Serum Gb3 recovered to lower levels after CsA treatment. Gb3 was undetected in wild-type liver, and the levels of Gb3 (but not gangliosides) in Fabry mouse liver were significantly depleted by CsA treatment. VT1 liver histochemistry showed Gb3 accumulated in Kupffer cells, endothelial cell subsets within the central and portal vein and within the portal triad. Hepatic venule endothelial and Kupffer cell VT1 staining was considerably reduced by in vivo CsA treatment. We conclude that MDR1 inhibition warrants consideration as a novel adjunct treatment for neutral GSL storage diseases.     

Acute kidney injury induced by protein-overload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides

Sulfatides, possible antithrombotic factors belonging to sphingoglycolipids, are widely distributed in mammalian tissues and serum. We recently found that the level of serum sulfatides was significantly lower in hemodialysis patients than that in normal subjects, and that the serum level closely correlated to the incidence of cardiovascular disease. These findings suggest a relationship between the level of serum sulfatides and kidney function; however, the molecular mechanism underlying this relationship remains unclear. In the present study, the influence of kidney dysfunction on the metabolism of sulfatides was examined using an established murine model of acute kidney injury, protein-overload nephropathy in mice. Protein-overload treatment caused severe proximal tubular injuries within 4 days, and this treatment obviously decreased both serum and hepatic sulfatide levels. The sphingoid composition of serum sulfatides was very similar to that of hepatic ones at each time point, suggesting that the serum sulfatide level is dependent on the hepatic secretory ability of sulfatides. The treatment also decreased hepatic expression of cerebroside sulfotransferase (CST), a key enzyme in sulfatide metabolism, while it scarcely influenced the expression of the other sulfatide-metabolizing enzymes, including arylsulfatase A, ceramide galactosyltransferase, and galactosylceramidase. Pro-inflammatory responses were not detected in the liver of these mice; however, potential oxidative stress was increased. These results suggest that down-regulation of hepatic CST expression, probably affected by oxidative stress from kidney injury, causes reduction in liver and serum sulfatide levels. This novel mechanism, indicating the crosstalk between kidney injury and specific liver function, may prove useful for helping to understand the situation where human hemodialysis patients have low levels of serum sulfatides.     

Glycosylceramide Synthesis in the Developing Spinal Cord and Kidney of the Twitcher Mouse, an Enzymatically Authentic Model of Human Krabbe Disease

Abstract: UDP-galactose:ceramide galactosyltransferase activity was assayed in the spinal cord and kidney of the recently discovered neurological mutant, the twitcher mouse, which is an enzymatically authentic model of human globoid cell leukodystrophy (Krabbe disease). The activity in the spinal cord was essentially normal during the early myelination period up to 15 days. There was a slight reduction at 20 days. At 25 and 33 days, the galactosyltransferase activity was drastically reduced compared to controls. In contrast, the galactosyltransferase activity in the kidney of twitcher mice remained normal throughout the developmental stages examined. Activity of the control enzyme UDP-glucose:ceramide glucosyltransferase was always normal in both the spinal cord and kidney. Thus, reduction of galactosylceramide synthesis occurs in the CNS secondarily to the pathological alteration of the oligodendroglia. No such reduction occurs in the kidney, at least for the last step of galactosylceramide synthesis. Reduced synthesis as the result of metabolic regulation in the presence of the catabolic block is therefore unlikely to be the cause of the lack of abnormal accumulation of galactosylceramide in the kidney of patients with globoid cell leukodystrophy.     

Protection from high fat diet-induced increase in ceramide in mice lacking plasminogen activator inhibitor 1

Obesity increases the risk for metabolic and cardiovascular disease, and adipose tissue plays a central role in this process. Ceramide, the key intermediate of sphingolipid metabolism, also contributes to obesity-related disorders. We show that a high fat diet increased ceramide levels in the adipose tissues and plasma in C57BL/6J mice via a mechanism that involves an increase in gene expression of enzymes mediating ceramide generation through the de novo pathway (e.g. serine palmitoyltransferase) and via the hydrolysis of sphingomyelin (acid sphingomyelinase and neutral sphingomyelinase). Although the induction of total ceramide in response to the high fat diet was modest, dramatic increases were observed for C16, C18, and C18:1 ceramides. Next, we investigated the relationship of ceramide to plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of plasminogen activation and another key player in obesity. PAI-1 is consistently elevated in obesity and thought to contribute to increased artherothrombotic events and more recently to obesity-mediated insulin resistance. Interestingly, the changes in ceramide were attenuated in mice lacking PAI-1. Mechanistically, mice lacking PAI-1 were protected from diet-induced increase in serine palmitoyltransferase, acid sphingomyelinase, and neutral sphingomyelinase mRNA, providing a mechanistic link for decreased ceramide in PAI-1-/- mice. The decreases in plasma free fatty acids and adipose tumor necrosis factor-alpha in PAI-1-/- mice may have additionally contributed indirectly to improvements in ceramide profile in these mice. This study has identified a novel link between sphingolipid metabolism and PAI-1 and also suggests that ceramide may be an intermediary molecule linking elevated PAI-1 to insulin resistance.     

Assessment of hepatoprotective and nephroprotective potential of withaferin A on bromobenzene-induced injury in Swiss albino mice: possible involvement of mitochondrial dysfunction and inflammation

Bromobenzene is a well-known environmental toxin which causes liver and kidney damage through CYP450-mediated bio-activation to generate reactive metabolites and, consequently, oxidative stress. The present study aimed to evaluate the possible protective role of withaferin A against bromobenzene-induced liver and kidney damage in mice. Withaferin A (10 mg/kg) was administered orally to the mice for 8 days before intragastric intubation of bromobenzene (10 mmol/kg). As results of this experiment, the levels of liver and kidney functional markers, lipid peroxidation, and cytokines (TNF-α and IL-1β) presented an increase and there was a decrease in anti-oxidant activity in the bromobenzene-treated group of mice. Pre-treatment with withaferin A not only significantly decreased the levels of liver and kidney functional markers and cytokines but also reduced oxidative stress, as evidenced by improved anti-oxidant status. In addition, the mitochondrial dysfunction shown through the decrease in the activities of mitochondrial enzymes and imbalance in the Bax/Bcl-2 expression in the livers and kidneys of bromobenzene-treated mice was effectively prevented by pre-administration of withaferin A. These results validated our conviction that bromobenzene caused liver and kidney damage via mitochondrial pathway and withaferin A provided significant protection against it. Thus, withaferin A may have possible usage in clinical liver and kidney diseases in which oxidative stress and mitochondrial dysfunction may be existent.     

Alteration of phospholipid composition of mouse liver microsomes in vivo and the effect on membrane properties

Administration of the methylation inhibitor periodate-oxidized adenosine to male Swiss-Webster mice on a choline-deficient diet produced a decrease (17%) in phosphatidylcholine to phosphatidylethanolamine ratios compared to saline-injected controls in liver, and also in kidney (11%), but not in muscle microsome preparations. Both intact liver microsomes and reconstituted membranes from lipid extracts showed a higher fluorescence anisotropy of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene than control samples in the temperature range of 20–31°C.     

Effects of swimming training on three superoxide dismutase isoenzymes in mouse tissues.

The purpose of the present study was to investigate the effects of swimming training on the changes in three superoxide dismutase (SOD) isoenzymes in mice. The trained mice underwent a 6-wk swimming program (1 h/day, 5 days/wk) in water at 35-36 degrees C. Immunoreactive extracellular SOD (EC-SOD), copper- and zinc-containing SOD (CuZn-SOD), and manganese-containing SOD (Mn-SOD) contents and their mRNA abundance were determined in serum, heart, lung, liver, kidney, and gastrocnemius muscle. EC-SOD content in liver and kidney was significantly increased with training. After training, CuZn-SOD content rose significantly only in kidney but decreased significantly in heart, lung, and liver. Mn-SOD content showed a significant increase in lung, kidney, and skeletal muscle but a significant decrease in liver. In most tissues, however, the changes in SOD isoenzyme contents were not concomitant with those in their mRNA levels. The results obtained thus suggest that, except for kidney, the responses in mouse tissues of three SOD isoenzymes (protein levels and mRNA abundance) to swimming training are different and that kidney may be one of the most sensitive organs to adapt to oxidative stress during physical training, although the mechanism remains vague.     

Glycolipid depletion in antimicrobial therapy

Mucosal pathogens target sites of infection through specific adherence to host glycoconjugate receptors. As a consequence, depletion of such receptors from the cell surface may be expected to inhibit attachment, impair bacterial colonization and reduce the activation of mucosal inflammation. We have used the glucose analogue and glycosphingolipid (GSL) biosynthesis inhibitor N-butyldeoxynojirimycin (NB-DNJ) to deplete human uroepithelial cells and the murine urinary tract mucosa of receptors for P-fimbriated Escherichia coli. NB-DNJ blocks the ceramide-specific glucosyltransferase, which catalyses the formation of glucosyl ceramide (GlcCer), the precursor for GSLs. The inhibitor was shown to decrease the GSL content in a dose-dependent way, and depletion markedly inhibited P-fimbriated bacterial attachment in vitro. NB-DNJ-fed C3H/HeN mice were depleted of GSLs in vivo and showed reduced susceptibility to experimental urinary tract infection with P-fimbriated E. coli. The mucosal inflammatory response was impaired, as shown by reduced chemokine secretion and lower neutrophil recruitment, and the bacteria colonized the urinary tract less efficiently than in normal mice. These results confirmed the role of P fimbriae-mediated adherence for colonization and inflammation and point to an interesting alternative to antibiotic treatment for urinary tract infection.     

Effects of potassium deficiency on potassium,polyamines and amino acids in mouse tissues

Sexual dimorphism in potassium content was found in plasma, kidney, heart and skeletal muscle of CD1 mice. We observed that feeding mice with a K(+)-deficient diet had an uneven and gender-dependent effect on organ weight and tissue potassium concentrations. Treatment produced a marked decrease in plasma, pancreas and skeletal muscle K(+) levels in both sexes, and a reduction in kidney, liver and heart potassium concentrations in females. Moreover, K(+) deficiency produced a 2-3-fold increase in the concentrations of cationic amino acids, such as arginine and lysine in both heart and skeletal muscle of the two sexes, a slight increase ( approximately 37%) in renal arginine in the male mice. The concentrations of these amino acids in plasma and other tissues in both sexes remained unaltered. Polyamine levels in heart, liver, skeletal muscle and pancreas from male and female mice were not affected by K(+) deficiency. However, in the male kidney potassium deficiency was accompanied by an increase of putrescine and spermidine concentration, and a reduction of putrescine excretion into the urine, even though renal K(+) concentration was not significantly affected and ornithine decarboxylase activity was dramatically decreased. The general lack of correlation between tissue potassium decrease and the increase in organic cations suggests that it is unlikely that the changes observed could be related with an attempt of the tissues to compensate for the reduction in cellular positive charge produced by the fall in K(+) content. The mechanisms by which these changes are produced are discussed, but their physiological implications remain to be determined.