Synthesis Of Lactosylceramide Research Articles

UGCG modulates heart hypertrophy through B4GalT5-mediated mitochondrial oxidative stress and the ERK signaling pathway

Mechanical pressure overload and other stimuli often contribute to heart hypertrophy, a significant factor in the induction of heart failure. The UDP-glucose ceramide glycosyltransferase (UGCG) enzyme plays a crucial role in the metabolism of sphingolipids through the production of glucosylceramide. However, its role in heart hypertrophy remains unknown. In this study, UGCG was induced in response to pressure overload in vivo and phenylephrine stimulation in vitro. Additionally, UGCG downregulation ameliorated cardiomyocyte hypertrophy, improved cardiomyocyte mitochondrial oxidative stress, and reduced the ERK signaling pathway. Conversely, UGCG overexpression in cardiomyocytes promoted heart hypertrophy development, aggravated mitochondrial oxidative stress, and stimulated ERK signaling. Furthermore, the interaction between beta-1,4-galactosyltransferase 5 (B4GalT5), which catalyses the synthesis of lactosylceramide, and UGCG was identified, which also functions as a synergistic molecule of UGCG. Notably, limiting the expression of B4GalT5 impaired the capacity of UGCG to promote myocardial hypertrophy, suggesting that B4GalT5 acts as an intermediary for UGCG. Overall, this study highlights the potential of UGCG as a modulator of heart hypertrophy, rendering it a potential target for combating heart hypertrophy.

B4GALT5 high expression associated with poor prognosis of hepatocellular carcinoma

BackgroundB4GALT5 is postulated to be an important protein in sugar metabolism that catalyzes the synthesis of lactosylceramide (LacCer). However, its role in hepatocellular carcinoma (HCC) remains unknown.MethodWe characterized the expression of B4GALT5 in HCC tissue compared to normal tissue, and explored its function of B4GALT5 in HCC by enrichment analysis based on its co-expressed gene set. Next, we checked whether B4GALT5 expression is correlated to immune infiltration level and clinical prognosis in hepatocellular carcinoma. Finally, we verified the expression of B4GALT5 using clinical samples evaluated by RT-PCR, and conducted in vitro experiments with B4GALT5-knockdown HCC cells to investigate the function of B4GALT5 in the HCC cell proliferation, migration and invasion.ResultsWe found B4GALT5 mRNA and protein expression levels were significantly high in HCC tissue compared to normal tissue. The enrichment analysis of the gene sets that co-expressed with B4GALT5 showed specificity in HCC-related pathways and functions. Also, the expression pattern of B4GALT5 was significantly related to the immune infiltration level, especially CD4+ T cell and macrophage cells. B4GALT5 higher mRNA expression was associated with poor overall survival (OS) in HCC patients. Furthermore, In vitro experiments showed that depletion of B4GALT5 significantly inhibited HCC cell proliferation, migration and invasion. This study revealed the function and its mediated pathways of B4GALT5 in HCC, indicating that B4GALT5 may serve as a prognostic biomarker of HCC.

B4GALT6 regulates astrocyte activation during CNS inflammation (INM8P.360)

Abstract Astrocytes play complex roles in neuroinflammation. Thus, it is important to characterize the mechanisms regulating astrocyte function, as well as potential targets for the therapeutic modulation of astrocyte activity. Here we report that during the chronic-progressive phase of EAE, astrocytes promote disease pathogenesis by a lipid-dependent signaling pathway. In line with previous studies, depletion of reactive astrocytes during the acute phase of chronic-progressive EAE exacerbated disease. However, depletion of astrocytes in the chronic phase ameliorated the disease. Hence, to understand this dichotomy we compared gene expression profiles of astrocytes from mice in acute and chronic stages of EAE. One gene associated with the chronic stage was B4GALT6, which codes for the β-1,4-galactosyltransferase 6 that catalyzes the synthesis of lactosylceramide (LacCer). We observed that LacCer levels are upregulated in the CNS during chronic EAE, and that LacCer synthesized by the astrocytes acts in an autocrine manner to trigger transcriptional programs that promote the recruitment and activation of infiltrating monocytes and microglia, and neurodegeneration. We also detected increased B4GALT6 expression and LacCer levels in MS lesions. Finally, the inhibition of LacCer synthesis suppressed CNS innate immunity and neurodegeneration, and interfered with the activation of human astrocytes. Thus, B4GALT6 is a potential therapeutic target for MS and neuroinflammatory disorders.

4GalT6 is involved in the synthesis of lactosylceramide with less intensity than 4GalT5

Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, β1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of β4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that β4GalT6 plays a role as a LacCer synthase, whereas β4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.

Accumulation of lactosylceramide and overexpression of a PSC833-resistant P-glycoprotein in multidrug-resistant human sarcoma cells

The selection pressure for resistance to chemotherapy is accompanied by the enhanced expression of ABC proteins and increased cellular glycosphingolipid content. Thus, a possible connection between glycosphingolipid metabolism and ABC proteins in drug resistance has been suggested. In the present study, we established two human multidrug-resistant (MDR) cell lines derived from MESSA sarcoma cells by culturing with increasing concentrations of doxorubicin (DX5 cells) or doxorubicin together with cyclosporin A (GARF cells). Both resistant cell lines overexpressed the MDR1 gene and the wild-type P-glycoprotein at the same level. The cyclosporin derivative PSC833, a potent inhibitor of P-glycoprotein, sensitized DX5 but not GARF cells to the cytotoxic effects of daunorubicin. Moreover, PSC833 increased the nuclear accumulation of daunorubicin and the cellular accumulation of [3H]vinblastine in the DX5 but not in the GARF cells. The cellular incorporation of [3H]-cyclosporin A was lower in DX5 cells compared to MESSA and GARF cells, which incorporated the same level of [3H]-cyclosporin A. Sphingolipid analysis showed that the lactosylceramide level was 2.5- and 5-fold higher in DX5 and GARF cells, respectively, than in MESSA cells. Whereas the pharmacological inhibition of lactosylceramide synthesis was able to reverse only partially the resistance of GARF cells to daunorubicin without significant increase in nuclear accumulation of the drug, the same treatment before the co-treatment with PSC833 and daunorubicin increased the cytotoxic effect of daunorubicin and its nuclear accumulation. These data suggest a possible relationship between lactosylceramide levels and the resistance of P-glycoprotein to modulation by MDR modulators.

Tumor Necrosis Factor‐[Alpha] regulates lactosylceramide synthesis via activation of Protein Kinase C: A novel function of Gi‐linked sphingosine‐1‐phosphate receptor

Tumor Necrosis Factor‐[Alpha] regulates lactosylceramide synthesis via activation of Protein Kinase C: A novel function of Gi‐linked sphingosine‐1‐phosphate receptor

The CLN9 Protein, a Regulator of Dihydroceramide Synthase

A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients.

Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum.

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.

Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

Multidrug-resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug-resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug-resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell-free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.

Glucosylceramide synthesized in vitro from endogenous ceramide is uncoupled from synthesis of lactosylceramide in Golgi membranes from chicken embryo neural retina cells.

It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[3H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[3H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [3H]GlcCer from those enriched in [3H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.

Functional Organization of the Golgi Apparatus in Glycosphingolipid Biosynthesis: LACTOSYLCERAMIDE AND SUBSEQUENT GLYCOSPHINGOLIPIDS ARE FORMED IN THE LUMEN OF THE LATE GOLGI

Biosynthesis of plasma membrane sphingolipids involves the coordinate action of enzymes localized to individual compartments of the biosynthetic secretory pathway of proteins. These stations include the endoplasmic reticulum and the Golgi apparatus. Although a precise localization of all the enzymes that synthesize glycosphingolipids has not been achieved to date, it is assumed that the sequence of events in glycosphingolipid biosynthesis resembles that in glycoprotein biosynthesis, i.e. that early reactions occur in early stations (endoplasmic reticulum and cis/medial Golgi) of the pathway, and late reactions occur in late stations (trans Golgi/trans Golgi network). Using truncated analogues of ceramide and glucosylceramide that allow measurement of enzyme activities in intact membrane fractions, we have reinvestigated the localization of individual enzymes involved in glycosphingolipid biosynthesis and for the first time studied the localization of lactosylceramide synthase after partial separation of Golgi membranes as previously described (Trinchera, M., and Ghidoni, R. (1989) J. Biol. Chem. 264, 15766-15769). Here, we show that the reactions involved in higher glycosphingolipid biosynthesis, including lactosylceramide synthesis, all reside in the lumen of the late Golgi compartments from rat liver.

GT3 synthesis in the proximal Golgi occurs in a compartment different from those for GD3 and GM3 synthesis.

Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [3H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[3H]Gal. In (a), 1 microM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to approximately 50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of > 90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited approximately 30 and 70%, respectively, in 7- and 10-day cells. In (b), > 80% of the [3H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [3H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, < 5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[3H]NeuAc incorporated approximately 20% of [3H]NeuAc into endogenous GT3, and this percentage was not affected by 1 microM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.

Oxidized low density lipoprotein stimulates aortic smooth muscle cell proliferation.

We have investigated the effects of oxidized low density lipoproteins (Ox-LDL) on aortic smooth muscle cell (SMC) proliferation and the biosynthesis of glycosphingolipids. We found that Ox-LDL exerted a concentration, time, and temperature dependent alteration of cell proliferation and the biosynthesis of lactosylceramide. At low concentrations (5-10 micrograms/ml medium) Ox-LDL stimulated cell proliferation measured by an increase in the incorporation of [3H]-thymidine in cells and the synthesis of lactosylceramide, but not glucosylceramide synthesis. Oxidized LDL exerted a threefold increase in the incorporation of [3H]-galactose and [3H]-serine in lactosylceramide. The activity of lactosylceramide synthetase; UDP-galactose glucosylceramide beta 1 --> 4 galactosyltransferase (GalT-2), but not glucosylceramide synthetase (GlcT-1) was stimulated by Ox-LDL. On the other hand, LDL suppressed the activity of GalT-2 in these cells. When cells were preincubated with antibody against Ox-LDL or GalT-2 it compromised the Ox-LDL mediated stimulated in cell proliferation and GalT-2 activity. Similarly, D-PDMP an inhibitor of GalT-2 compromised the Ox-LDL mediated effects in cells. In contrast, L-PDMP further stimulated the Ox-LDL mediated cell proliferation and GalT-2 activity. However, preincubation of cells with preimmune rabbit serum IgG failed to abrogate Ox-LDL mediated stimulation in cell proliferation and GalT-2 activity. In sum, we found that Ox-LDL stimulated aortic smooth muscle cell proliferation in culture. This effect resulted from Ox-LDL mediated activation of GalT-2 that produced lactosylceramide. Lactosylceramide in turn, contributed to cell proliferation. Such correlations are supportive of the notion that GalT-2 action mediates the signal transduction of Ox-LDL contributing to cell proliferation.

Lactosylceramide is synthesized in the lumen of the Golgi apparatus

Recently, synthesis of lactosylceramide has been described to occur on the cytosolic face of the Golgi [(1991) J. Biol. Chem. 266, 20907-20912]. The reactions following in the biosynthesis of higher glycosphingolipids are known to take place in the lumen of the Golgi. For our understanding of the functional organization of the multiglycosyltransferase system of glycosphingolipid synthesis in the Golgi, the knowledge of the topology of individual reactions is a prerequisite. We have developed a simple and quick assay system for sphingolipid biosynthesis and have obtained evidence that lactosylceramide is synthesized in the lumen of the Golgi. Because lactosylceramide is generated by galactosylation of glucosylceramide which, in turn, is synthesized from ceramide and UDP-Glc on the cytosolic surface of the Golgi apparatus, further efforts will be directed to the characterization of a glucosylceramide-translocator in the Golgi membranes rather than a lactosylceramide-translocator.

Regulation of synthesis of lactosylceramide in normal and tumor proximal tubular cells

We have measured the binding and degradation of low-density lipoprotein (LDL) and LDL-mediated effects on cholesteryl ester (CE) synthesis in cultured normal and tumor proximal tubular (PT) cells. The effects of LDL on the regulation of glycosphingolipid metabolism in cells was pursued employing radioactive precursors, e.g., [ 3H]serine, and [ 3H]glucose and by measuring the activity of UDP-galactose:glucosylceramide:B1-4 galactosyltransferase (GalT-2). In normal PT cells, there was a saturable and displaceable binding and degradation of 125I-LDL and a LDL mediated 14-fold stimulation of cholesteryl ester (CE) synthesis. This was accompanied by a suppression (70–80%) of incorporation of [ 3H]glucose and [ 3H]serine into GlcCer, LacCer, GbOse 3Cer and GbOse 4Cer and suppression (70–80%) of GalT-2 activity. In tumor PT cells, displaceable binding and degradation of 125I-LDL was not observed and LDL failed to stimulate CE synthesis. In such cells, LDL exerted a concentration-dependent stimulation of [ 3H]glucose and [ 3H]serine incorporation into GSL. Maximum stimulation (250%) of GalT-2 activity in tumor PT cells occurred with 25 μg LDL/ml medium. We conclude that LDL taken up via receptor mediated pathway decreases GaIT-2 activity in normal PT cells. In contrast, LDL not taken up via the LDL receptor pathway in tumor PT cells failed to suppress the incorporation of [ 3H]glucose and [ 3H]serine into glycosphingolipids and GalT-2 activity leading to a stimulation of lactosylceramide synthesis.

Regulation of glycosphingolipid glycosyltransferase by low density lipoprotein receptors in cultured human proximal tubular cells.

We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis.

Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells.

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.

Synthesis of double-labeled lactosylceramide

The total stereo-controlled synthesis of lactosylceramide and introduction of two kinds of isotopes,3H and14C, into synthetic lactosylceramide are described.

Enzymatic synthesis of lactosylceramide by a galactosyltransferase from developing chicken retina

The activity of UDP-galactose: glucosylceramide galactosyltransferase was studied during chick retina ontogenesis. This galactosyltransferase catalyses the formation of lactosylceramide, a reaction which is considered to be one of the initial steps of ganglioside biosynthesis. The activity was detected in 7 day-old embryos and was present in adult animals. The highest specific activity was found at the 11th day of embryonic life. The radioactive product was identified as lactosylceramide by comparison with standard glycolipids, by radiochromatography, by thin layer chromatography of the o-methyl-glycoside derivatives and by gas-liquid chromatography. The enzyme required the presence of Mn 2+, had a pH optimum of about 6·7 and was stimulated by Triton X-100.

Alterations of glycolipid glycosyltrasnferase levels in lymphoma and lymphoid tissues of lymphoma-bearing hamsters.

Thymus from lymphoma-bearing hamsters in advanced stages of tumor growth showed enhanced activities of four glycolipid-glycosyltransferases which lead to the step-wise synthesis of lactosylceramide to Forssman hapten. The alpha-galactosyltransferase activity of the thymus was decreased until day 6 of inoculation but increased thereafter. The activities of all the transferases tested were significantly lowered in lymphoma tissue. The level of UDP-galactose pyrophosphatase did not correlate with the transferase activities.