Institute of Clinical Neuroscience, Section of Experimental Neuroscience, Göteborg University, Sahlgrenska University Hospital/Mölndal, SE-431 80 Mölndal, Sweden, 2Bartholin Instituttet, Kommunehospitalet, DK-1399 Copenhagen K, Denmark, 3Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institute, SE-171 77 Stockholm, Sweden, and 4Department of Pathology, Herlev Hospital, University of Copenhagen, DK 2730 Herlev, Denmark
Received on March 27, 1999; revised on July 5, 1999; accepted on July 22, 1999.
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Abstract |
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Key words: glycosphingolipids/insulin/metabolism/recycling/sulfatide
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Introduction |
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The presence of sulfatide in pancreatic tissue and in isolated islets of Langerhans was consistent among various species including man, rat, mouse, pig, and monkey (Buschard et al., 1993bb, 1994). Immunohistochemical analyses of pancreatic tissue using the anti-sulfatide monoclonal antibody, Sulf I (Buschard et al., 1993b
b, 1994), revealed selective staining in the islets of Langerhans, whereas the exocrine tissue was unlabeled. Biochemical analyses of pancreatic tissue and isolated
and ß cells confirmed that among potential glycolipid antigens (sulfatide, sulfated lactosylceramide and seminolipid) sulfatide was the only antigen expressed (Buschard et al., 1994
). Moreover, immune electron microscopy showed that sulfatide was present in secretory granules (Buschard et al., 1993b
b).
During the last few years it has become clear that membrane-bound lipids play a role in endocytosis (for review, see Riezmann et al., 1997) and in intracellular vesicle trafficking and sorting (van Meer, 1998). Among the these lipids are the glycosphingolipids, which have attracted special interest as components in caveolae (Parton, 1996
; Simons and Ikonen, 1997) and in forming domains with specific proteins (Kasahara et al., 1997
). Sulfatide is synthesized through a stepwise enzymatically catalyzed process starting in the endoplasmic reticulum and finally being sulfated in the Golgi (Benjamins et al., 1982
), presumably the late Golgi or trans-Golgi network (Farrer et al., 1995
), from which it is transported by membrane vesicle flow to the plasma membrane and possibly other intracellular organelles, like the lysosome. The presence and specificity of proteins in such vesicle remains an open question.
The production of insulin starts in the endoplasmic reticulum with the synthesis of pre-proinsulin, which is converted to proinsulin and then transferred to the Golgi (for review, see Orci, 1986). It is also known that proinsulin is released from the trans-Golgi network in secretory granules. During the cytoplasmic transport of these secretory granules from the Golgi to a location close to the plasma membrane the proinsulin is transformed to insulin. Sulfatide and insulin thus both seem to be processed through the trans-Golgi network compartment and possibly sorted into and transported in the same granulae.
Based on these previous findings together with immune electron microscopy investigations showing that sulfatide, like insulin, is present in secretory granules (Buschard et al., 1993b), we hypothesized that there might be a functional association between sulfatide and insulin, possibly related to the intracellular trafficking of secretory granulae and/or insulin processing.
The aim of this study was to find evidence for synthesis of sulfatide in the islets of Langerhans, and if it exits, to elucidate the metabolic pathway of sulfatide in these cells. The study was performed using freshly isolated rat islet cells in culture and metabolic labeling with endogenous 35S-sulfate, 3H-galactose, and 14C-serine in the presence or absence of various inhibitors, Brefeldin A, fumonisin B1 and chloroquine, each described to inhibit different processes involved in synthesis of glycosphingolipids. The results showed that sulfatide was produced in the cells and that the main route of synthesis was recycling, where sulfatide was partially degraded in the lysosomes and then reutilized for synthesis in the Golgi. Together with the findings that sulfatide is present in the insulin secretory granules and that a major fraction of the insulin is degraded through the lysosomes (Halban and Wollheim, 1980), this lends support to a biologically relevant association between sulfatide and insulin in the cellular processing of insulin in islet cells in rat pancreas.
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Results |
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TLC-ELISA using the SulfI antibody showed two stained bands migrating (Figure 1) in the same region as the sulfatide standard isolated from pig brain. There were no stained bands migrating similarly to the other antigens, seminolipid and sulfated lactosylceramide. Acid hydrolysis of the sulfatide fraction abolished all staining with the antibody. The endogenous sulfatide content varied between the experiments from 3 to 5 nmol/mg protein. No difference was found between cells grown in 2.8, 11, and 20 mM glucose.
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The mass spectra of Lewis rat brain sulfatide (Table I) showed that major molecular species contained the fatty acids C24:0 and C24:1 and also the corresponding hydroxy fatty acids. In the islet cells, the two dominating molecular species of sulfatide contained C16:0 and C24:0 fatty acids. Other prominent peaks corresponded to fatty acids C22:0 and C24:1. All molecular species contained 4-sphingenine as long chain base. Thus, in comparison with sulfatide isolated from rat brain, the islet cell sulfatide had a higher proportion of short chain fatty acids and in addition no measurable amounts of hydroxy fatty acids. No difference in the fatty acid composition of sulfatide from islet cells cultured in 2.8, 11, or 20 mM glucose medium was found.
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Determination of mRNA for UDP-galactose:ceramide galactosyltransferase and sulfotransferase in isolated rat islets cells
mRNA for UDP-galactose:ceramide galactosyltransferase was detected in islet cells and the glucose level in the medium had a direct effect on its expression (Figure 3). When hybridized to actin and normalized with this signal, the expression was 2.6 ± 0.64 (n = 5) more abundant (p = 0.0028, Mann Whitney U-test) in glucose stimulated (20 mM) than in glucose-deprived (2.8 mM) islets. There was no detectable sulfotransferase when using the cDNA probe provided by Dr. Honke (Honke, 1997).
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The effect of Brefeldin A as observed by adding 14C-serine (Figure 4A) was a 3-fold increase of c.p.m. in the sphingomyelin fraction and a 6-fold increase of ceramide, which was identified by HPTLC and autoradiography. Labeling with 35S-sulfate (Figure 4C) was abolished in the presence of Brefeldin A, indicating that the recycling pathway was affected.
Effect of Fumonisin B1 on the metabolism of sulfatide in islets cells
Incubation of the islet cells in medium with Fumonisin B1 for 40 h markedly inhibited the incorporation of 14C-serine (Figure 5A). Two weak bands were seen of which the major one migrated similarly to sphingomyelin. However analyses on borate-impregnated plates showed that at most half the radioactivity corresponded to sphingomyelin. No further characterization was performed.
In the presence of Fumonisin B1 the 3H-galactose labeling of sphingolipids was (Figure 5B) was reduced. The incorporation of 35S-sulfate was also inhibited, as revealed by a reduced amount of labeled sulfatide. Shorter incubation, 6 h, with Fumonisin B1 (Figure 6) had no effect on the 35S-sulfate labeling of sulfatide. These findings indicate that the effect of Fumonisin B1 on the recycling pathway is a later event and most likely a secondary effect of the inhibition of de novo synthesis of sphingolipids.
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Discussion |
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The study was performed on isolated islets from rat pancreas which contain approximately 75% insulin-producing ß-cells. The structure of the sulfatide present in the islets was confirmed but showed a fatty acid composition that was different as compared to sulfatide in the brain of Lewis rats and other mammalian species (Norton and Cammer, 1984; Vos et al., 1994; Ishizuka, 1997
). The most striking difference was the high proportion of the short fatty acid C16:0 and the lack of hydroxy fatty acids. Similar results were recently obtained (Hsu et al., 1998
) when analyzing islet cells isolated from pancreas from SpragueDawley rats. A relatively high proportion of nonhydroxylated fatty acids has also been reported to occur in sulfatide from mammalian kidney (Ishizuka, 1997
).
The fatty acid chain length, hydroxylation and degree of unsaturation affect the physicochemical properties of the molecule in the membrane (Stevensson et al., 1992; Tupper et al., 1992
; Vos et al., 1994
; Ishizuka, 1997
). Sulfatide in myelin contains more saturated or monounsaturated long fatty acids (C22C26), both with and without 2-hydroxy groups, as compared to most other sphingolipids and phospholipids and spans more than half the membrane lipid bilayer and interdigitates into the opposite monolayer. The large proportion of shorter chain length (C16:0) fatty acid in sulfatide from islets cells does thus not have such interdigitation properties and lateral migration in the membrane is therefore facilitated. It is beyond the scope of the experiments done so far to identify the precise role of the short fatty acid chains in the sulfatide molecule in the islets, but they show that sulfatide in these cells has different physicochemical properties than sulfatide in myelin. This in turn indicates that this molecule might have different functions in islets and myelin. It is also likely that molecular species with shorter and longer fatty acids have a different distribution and function in the insulin-producing cells.
Another physicochemical property of sulfatide is that these molecules might associate and form patches in the membranes, a phenomenon that has been suggested to support close contact between membranes, as in fusion processes (Hakomori, 1991). Sulfatide is also a strong Ca2+ binder and binding induces a hydrocarbon chain disorder preferentially in nonhydroxylated molecular species. It is therefore tempting to speculate that the lack of hydroxylated fatty acids in islet cells favors its involvement in membrane fusion being influenced by Ca2+ alterations, which precede glucose-induced insulin secretion. Another effect of the fatty acid composition is that it influences the metabolic pathway (van Meer, 1998).
Based on previous results, it is generally assumed that sulfatide is formed by sulfation of galactosylceramide (Vos et al., 1994; Farrer et al., 1995
; Ishizuka, 1997
). Farrer et al. (1995)
showed that addition of Brefeldin A led to almost complete cessation of sulfatide synthesis in immortalized Schwann cells and, as in this study, inhibition was reached after 6 h treatment with 0.5 µg Brefeldin A/ml. Brefeldin A is known to mainly affect the transport between the ER and cis/medial-Golgi (reviewed by Klausner et al., 1992
) and inhibits glycosylation of complex glycosphingolipids (van Echten et al., 1995
) and sulfatide (Farrer et al., 1995
) which takes place in the trans-Golgi network. The results obtained in this study are in conformity with the described effect of Brefeldin A. Brefeldin A has also been shown to inhibit glycosylation of recycled glycosphingolipids (Gordon and Lloyd, 1994
), which would then affect the major pathway for sulfatide synthesis in the islets of Langerhans. Moreover, Huang and Arvan (1994)
showed that Brefeldin A inhibited the transfer of pro-insulin from the trans-Golgi network to granules. If sulfatide, as we hypothesize, is sorted into the same granules as pro-insulin the inhibited sulfatide synthesis might reflect the lack of pro-insulin granule formation.
In addition to the inhibition of sulfatide synthesis, Brefeldin A was found to increase glucosylceramide and lactosylceramide formation. This is in contrast to the study by Farrer et al. (1995), who instead found a concomitant increase of galactosylceramide. However, they studied another cell type, immortalized Schwann cells and glycosphingolipid synthesis is known to be cell type and cell differentiation specific (Hakomori, 1981). Brefeldin A was also found to induce a significant increase of formation of sphingomyelin and ceramide in the rat islets. The increase of cellular ceramide might be one factor that induced the disintegration seen in the islets of Langerhans when the incubation was prolonged for more than 6 h. Ceramide has been shown in many studies to induce apoptosis (Merill et al., 1997
) and the 6-fold increase is likely to induce such a process.
Fumonisin B1 is a fungal toxin that inhibits the acylation of sphingosine (sphingenine) by acylCoA to form ceramide and thereby the de novo synthesis of all products produced from ceramide, including sphingomyelin and glycosphingolipids. The weak labeling obtained with 3H-galactose might reflect the fact that the inhibition was not complete and/or that there was a re-use of partially degraded sphingolipid products from the recycling pathway. Although the latter pathway is not primarily affected by fumonisin, a marked reduction of 35S-sulfate-labeled sulfatide was seen after 40 h exposure. This might reflect the fact that during a 40-hour period the de novo synthesis is required to restore the endogenous pool and/or that the effect of fumonisin in the long run has a general effect on cellular metabolism. Islets exposed to fumonisin for only 6 h had no effect on 35S-sulfate-labeled sulfatide, supporting that recycling is the dominating pathway.
The third glycosphingolipid inhibitor used in this study was chloroquine. This compound is an amphiphilic molecule known to accumulate in the lysosome (DeDuve et al., 1976) and possibly in the early endosomes, where it causes a rise in pH, which in turn effect the activity of the lysosomal enzymes having acidic pH optima. The inhibitory effects of chloroquine on lysosomal degradation of glycosphingolipids was reported many years ago (Klinghardt et al., 1981
). The inhibitory effect of chloroquine on the 35S-sulfate labeling of sulfatide might thus be explained by a reduced activity of the sulfohydrolase in the lysosome and thereby reduced formation of product for the recycling pathway of sulfatide synthesis.
Finally, we looked into the effect of a monoclonal antibody to sulfatide (Sulf I) with the aim of influencing recycling of sulfatide localized to the cell surface. However, there was no detectable reduction in 35S-sulfate-labeled sulfatide supporting that the main route for sulfatide biosynthesis is recycling of intracellularly localized sulfatide, i.e., in the secretory granules.
The reason for using 3H-galactose in the labeling studies was to explore whether the recycling involved just desulfation or in addition removal of galactose to form ceramide. However, the incorporation of galactose was in most experiments too low to permit any firm conclusions. The most common way to increase labeling with exogenous substrate is to exclude the native molecule from the incubation medium. However, as the metabolism of islets is regulated by exogenous glucose, it is likely that the uptake of glucose competes with that of galactose and removal of glucose would create a nonphysiological environment. Biochemical analyses revealed the existence of endogenous galactosylceramide but the main route of synthesis remains to be resolved.
The endogenous pool of insulin is large and a substantial fraction of insulin is never secreted but degraded within the ß-cell (Halban and Wollheim, 1980). Insulin-containing granules are fused with primary lysosomes and completely degraded. Sulfatide has previously been shown to be expressed in the secretory granules (Buschard et al., 1993b
, 1994), and in this study also in Golgi and lysosomes. Some lysosomes were intensely labeled whereas others were not labeled at all. The varying labeling intensity of lysosomes might reflect different steps in the degradation of sulfatide and insulin. These findings support a fusion of sulfatide-containing secretory granules with lysosomes.
There was no measurable change in the endogenous amount or the pulse chase labeling of sulfatide due to glucose levels in the media. As sulfatide is also located in the secretory granules, it is reasonable to assume that an increased secretion will reduce the proportion of secretory granula that enter the lysosome and thereby reduce the major pathway, recycling, for sulfatide synthesis. There was, however, increased sulfatide labeling in the lysosomes in islets stimulated with high glucose concentration, a finding that does not necessarily reflect increased fusion of secretory granules, but perhaps reduced degradation in stressed ß-cells. High glucose levels (high glucose) might have induced a de novo synthesis of insulin (matured from pro-insulin, see below) which is not immediately followed by a corresponding increase of de novo sulfatide synthesis. This is supported by recent data (Buschard et al., in press) showing that the number of secretory granules expressing sulfatide is more rapidly reduced than the total number of insulin granules.
It was noticed, however, that the mRNA level of the UDP-galactose:ceramide galactosyltransferase (CGT) (Stahl et al., 1994; Coetzee et al., 1996
) was significantly increased in islets grown in high glucose medium. This finding indicated that, at least after 24 h, there was a need for increased synthesis of galactosylceramide, which is the immediate precursor of sulfatide.
The most relevant enzyme mRNA to measure would have been 3'-phosphoadenylsulfate:galactosylceramide 3'-sulfotransferase. The only potential cDNA described is that found by Honke et al. (1997). Their report clearly showed that the gene encodes a lipid sulfotransferase but the acceptor(s) was not characterized. The exact specificity remains to be clarified. However, we could not detect any mRNA for this enzyme when using the probe kindly provided by Dr. Honke. One possible explanation is that this probe was made from a human renal cancer cDNA and there might be sequence differences between humans and rats. Another possibility might have been to measure the cell-free activity of the corresponding sulfotransferase but such analyses includes the use of detergent and other factors to optimize the milieu and might thus not reflect the cellular activity, which was the aim of this study.
In conclusion, this study has provided evidence that sulfatide is produced in islets of Langerhans and that the fatty acid composition, and thereby the physicochemical properties, differ from those of sulfatide in myelin. The main pathway of sulfatide synthesis was recycling, involving partial degradation of sulfatide in the lysosome. The presence of both sulfatide and insulin, in the Golgi, in secretory granules and in the lysosomes, and the fact that most of the insulin is degraded without ever being released, indicates that insulin and sulfatide take the same intracellular route and supports a functional association of insulin and sulfatide. Based on these results, we have raised the hypothesis that sulfatide might play a role in the trafficking of in islets of Langerhans from rat pancreas secretory granules and possible also in the processing of insulin.
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Materials and methods |
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14C-Labeled sulfatide and galactosylceramide, used as standards in autoradiography, were prepared in our laboratory. Briefly, sulfatide or galactosylceramide was deacetylated in alkaline methanol (Neuenhofer et al., 1985). The formed lysocompound was reacetylated with 14C-labeled palmitoylchloride in 50% sodium acetate (Karlsson et al., 1990
). 3H-Labeled sulfatide and galactosylceramide were prepared according to the procedure described by Schwarzmann (1978)
. The radiolabeled compounds were purified by preparative silica gel column chromatography and thin layer chromatography.
The materials used for RNA isolation and Northern blots were the following: Genescreen Plus membrane from NEN (Boston); Elutip from Schleucher and Schuel, Germany; 32P (dCTP)from Amersham, Bucks, England; Sephadex G50 from Pharmacia, Uppsala, Sweden, and reflection film and intensifying screen from NEN. LR-white, used for electron microscopy, was obtained from Bio-Rad, Watford, UK. The phosphatase substrate 5'-bromo-4'-chloro-3'indolylphosphate was purchased from Sigma, St. Louis, MO.
Antibodies
The production and characterization of the mouse monoclonal antibody Sulf I has been reported previously (Fredman et al., 1988). The glycosphingolipid antigens recognized have been shown to be sulfatide, lactosylceramide sulfate and seminolipid (Buschard et al., 1994
), but not sulfated glucosylceramide (Iida et al., 1989
). Alkaline-phosphatase-conjugated goat anti-mouse Ig antibody was purchased from Jackson laboratories (West Grove, PA), the rabbit anti-mouse Ig (F261) from Dako (Copenhagen, Denmark) and the anti-rabbit immunoglobulin conjugated with colloidal gold from Amersham. As control antibody to Sulf I the mouse anti-human CD8 (IgG1) antibody from Dako (M707) was used. The hybridoma-producing anti-gaclactosylceramide monoclonal antibody, O1 (Sommer and Schachner, 1981
; Bansal et al., 1989
) (mouse IgM) was kindly provided by Dr. Steve Pfeiffer, Department of Microbiology, University of Connecticut, Farmington, CT.
Isolation of islet cells from rat pancreas
Islets of Langerhans from 8-week-old male Lewis rats (purchased from Möllegård, LI Stensved, Denmark) were isolated under sterile conditions by a collagenase digestion technique described previously (Buschard et al., 1990). Isolated islets were suspended in RPMI 1640 (Gibco), containing 11 mM glucose, 10% fetal calf serum, 1% penicillinstreptomycin (10,000 IU/ml-10,000 µg/ml, Gibco, Paisley, UK) and with the pH adjusted to 7.35. The islets were incubated for ~20 h at 37°C and thereafter used for the various experiments described below. Usually, about 2000 islets could be isolated on each occasion, using four to six rats. Since the precise number of cells per islet and the actual condition of the cells may vary from one isolation to another, a direct comparison can only be made on islets isolated on the same occasion.
Growth of islets for preparation of sulfatide for determination of the ceramide composition
Islets (~2000) were after 20 h in 11 mM glucose medium, divided into three equal portions and transferred to medium containing 2.8, 11, or 20 mM glucose for 24 h. Extraction and isolation of sulfatide from two experiments (~4000 islets) was performed as described below. The lipid extracts were analyzed with TLC-ELISA. Sulfatide fractions for mass spectrometry analysis were isolated by preparative HPTLC using C/M/W (65:25:4, by vol.) as the developing solvent. Bands with migration corresponding to reference pig brain sulfatide were scraped out and extracted from the gel with C/M/W (3.6:2, by vol.) The mass spectra were recorded in the negative mode on a double-focusing magnetic mass spectrometer (Autospec, Micromass, Manchester, UK) equipped with en electrospray ionization source (Ghardashkhani et al., 1995) Part of the isolated sulfatide fractions was also subjected to acid methanolysis and analyzed on borate-impregnated HPTLC plates as described below.
In vitro biosynthesis of sulfatide in islets cells grown in high and low glucose medium
The cells were isolated and grown in 11 mM glucose for 20 h as described above. After washing, the cells were incubated for another 24 h in RPMI 1640 (Gibco) with supplements as described above but with a reduced glucose content, from 11 to 2.8 mM. Thereafter, the cells were randomly divided into equal portions and grown in medium with 2.8 (low glucose) or 20 mM glucose (high glucose). To the media was added 1 µCi/ml (final concentration) 14C-serine and/or 10 µCi/ml 35S-sulfate. After 24 h the cells were washed twice in ice-cold phosphate-buffered saline, PBS, and frozen at 80°C until biochemically analyzed. Approximately 500 islets were used for each assay and two independent assay set-ups were analyzed. The sphingolipid synthesis was analyzed by autoradiography and in some experiments scintillation counting was performed on gel fractions scraped out from the TLC plate and corresponding to individual glycosphingolipids.
Effect on the metabolism of sulfatide in islets cells in the presence of Brefeldin A
Islets were isolated and grown for 20 h in 11 mM glucose medium as described above. The islets were then divided into equal portions (the number of islets in each experiment being ~500) and grown in the same medium supplemented with 14C-serine (1 µCi/ml), 3H-galactose (2 µCi/ml) or 35S-sulfate (10 µCi/ml) and without or with Brefeldin A (0.5 or 5.0 µg/ml) for 6 h. (The reduced time of incubation as compared to the other experiments was due to the fact that the cells started to disintegrate after this time.) The islets were washed twice in ice-cold phosphate-buffered saline, PBS, and frozen at 80°C until biochemically analyzed. The experiment was performed on two occasions. The effect on sulfatide synthesis was analyzed by autoradiography and a scintillation count performed on gel fractions with individual glycosphingolipids.
Effect of Fumonisin B1 on the metabolism of sulfatide in islets cells
The experiments were performed as described for the Brefeldin A experiments, but with another incubation schedule. After the initial 20 h in 11 mM glucose medium fumonisin B1 (18.5 µg/ml) was added and the islets incubated for another 16 h. Thereafter, new medium (with 18.5 µg Fumonisin B1/ml) containing 14C-serine (1 µCi/ml), 3H-galactose (2 µCi/ml) or 35S-sulfate (10 µCi/ml) was added and the cells grown for another 24 h. The experiment was performed on two occasions. To elucidate the short-term effect, one experiment, in duplicate, was performed where islets after the first 20 h in 11 mM glucose medium were transferred to medium (11 mM glucose) without or with Fumonisin B1 (18.5 µg/ml) incubated for 1 h, after which 35S-sulfate was added and incubation proceeded for 5 h. The effect on sulfatide synthesis was analyzed by autoradiography and scintillation counting performed on gel fractions with individual glycosphingolipids.
Effect on the metabolism of sulfatide in islets cells in the presence of chloroquine
The islets were isolated and grown in 11 mM glucose medium for 20 h as described above. The islets were then divided into four fractions (~500 islets per fraction), two of which were used as controls and two were grown in the presence of chloroquine (25 µg/ml). The experiment was performed on two occasions. The incubation medium used was, as described above, supplemented with 20 mM glucose and 14C-serine (1 µCi/ml) or 35S-sulfate (10 µC/ml). The effect of chloroquine on the metabolic labeling during 6 h was analyzed by autoradiography and scintillation counting performed on gel fractions with individual glycosphingolipids.
Effect on the metabolism of sulfatide in islets cells in the presence of Sulf I antibody
Islet cells were isolated and grown in 11 mM glucose medium for 20 h as described above. Thereafter, the cells were incubated for 1 h in medium containing 20 mM glucose and the anti-sulfatide antibody Sulf I (40 µg/ml). Thereafter, without changing the media, 35S-sulfate (1 µCi/ml) was added. The effect of exogenously added antibody to sulfatide was analyzed by autoradiography of formed sphingolipids and scintillation counting performed on gel fractions with individual glycosphingolipids.
Extraction of lipids
The extraction and separation of the lipid fractions were performed as previously described (Buschard et al., 1993bb). Briefly, the lipids were extracted from the islet cells by homogenization in C/M/W (4:8:3, by vol.). The pellet was reextracted and the two supernatants combined. This total lipid extract was evaporated to dryness and redissolved in 1 ml of C/M/W (65:25:4, by vol.).
The lipids were then separated into two fractions by silica gel chromatography. Sulfatide, neutral monohexosylceramides, including galactosyl- and glucosylceramide, sulfated lactosylceramide and neutral glycosphingolipids with up to four sugar residues were eluted with 10 bed volumes of C/M/W (65:25:4, by vol.). This fraction, named the "sulfatide fraction" also contained ceramide and sphingomyelin and other lipids like fatty acids, cholesterol and phospholipids. To perform HPTLC- or TLC-separation for subsequent autoradiography and ELISA, respectively, the fraction had to be saponified (see below) to degrade phospholipids as some of these comigrated with the glycosphingolipid fractions and hampered the interpretation of the analyses.
Saponification of the sulfatide and galactosylceramide fraction
Saponification of the sulfatide fraction to degrade phospholipids was performed in the following way: an aliquot of the fraction was evaporated to dryness and redissolved in 200 µl of methanol/1 M KOH (1:1, v/v) and kept at room temperature over night. The samples were then neutralized with 0.5 M HCl and desalted on 2 x 0.5 g Sephadex G-25 (Wells and Dittmer, 1963).
Autoradiography and scintillation counting
Aliquots (corresponding to 50100 µg protein) of the lipid fractions were applied as 8 mm lanes to alumina-backed HPTLC plates and chromatographed in C/M/W (65:25:4, by vol.) The plates were air dried, sprayed with EnHance spray and exposed to x-ray film for 10 days. The autoradiogram was used to localize the individual glycosphingolipid fractions on the plate and the silica gel from these regions was scraped off and mixed with 10 ml scintillation liquid (Ultima Gold) and the samples counted in a Packard Tri-Carb 1500 Liquid Scintillation Analyzer.
Separation of isomeric monohexosylceramides (galactosyl- and glucosylceramide)
Isomeric monohexosylceramides comigrate on TLC and HPTLC plates in most organic solvents. Borate-impregnated plates on the other hand allow separation of isomeric hexaosylceramides. Briefly, HPTLC plates, were moistened by spraying them with 1% disodium-tetraborate-thiohydrate, Na2B4O7 x 10 H2O. The plates were air-dried for 4 h, and then dried further at 40°C in an oven for 16 h and kept in a desiccator until used. The chromatogram was developed with a solvent mixture of C/M/W (75:25:3, by vol.). The chromatography was repeated once in the same but newly prepared solvent. The plate was allowed to air-dry between runs. Autoradiography was performed as described above.
Separation of ceramide and free fatty acids
Ceramide and free fatty acids co-migrate in the solvents generally used in this study. To allow these molecules to be separated, the HPTLC plate was chromatographed in a solvent consisting of C/M/2.5M NH4 (90:10:1, by vol.). Autoradiography was then performed as described above.
Determinations of endogenous content of sulfatide and galactosylceramide in the islets
Sulfatide was quantified by TLC-ELISA using the sulfatide-specific SulfI monoclonal antibody (Fredman et al., 1988). Purified sulfatide standards and aliquots of the saponified sulfatide and galactosylceramide-containing lipid fractions from islets cells were applied as 5 mm lanes to plastic-backed HPTLC plates and chromatographed in C/M/W (65:25:4, by vol.). The plates were then incubated in sequential order with the Sulf I antibody, alkaline-phosphatase-conjugated anti-mouse antibody, and phosphatase substrate, 5'-bromo-4'-chloro-3'indolylphosphate. Galactosylceramide was determined with the same method using the O1 antibody for detection and standards of galactosylceramide.
Hydrolysis of the sulfate group from sulfatide
This analysis was performed on 14C-serine or 35S-labeled fractions from individual experiments. An aliquot of the saponificated sulfatide/galactosylceramide-containing lipid fractions from islet cells was evaporated to dryness. Sulfate was removed by incubating these fractions dissolved in 200 µl 0.05 M HCl in methanol at room temperature over night. The fractions were directly applied to alumina-backed HPTLC plates for autoradiography. Formed products were identified by their migration in relation to labeled sulfatide and galactosylceramide standards on HPTLC-plates. The chromatographic solvent used was C/M/W (65:25:4,by vol.). To discriminate between galactosyl- and glucosylceramide, borate-impregnated plates were used as described above.
Determination of UDP-galactose:ceramide galactosyltransferase mRNA in rat islets cells
Islets were isolated and then incubated for 20 h in RPMI 1640 containing 11 mM glucose as described above. Thereafter, the cells were divided into two portions and transferred to medium with 2.8 mM or 20 mM glucose, respectively. After another 24 h, the cells were lysed in guanidinium thiocyanate-phenol-chloroform (Chomczynski and Sacchi, 1987). An aliquot corresponding to 10 µg of total RNA was electrophoresed in 3-[N-morpholino] propanesulfonic acid-buffered agarose gel containing 0.66 M formaldehyde. The RNA was then transferred to Genescreen Plus membrane (NEN Dupont, Boston, MA). Five independent experiments were performed.
A fragment of the cDNA clone coding for UDP-galactose:ceramide galactosyltransferase containing the entire 2.4 kb insert was excised by BstxI digestion, isolated by agarose electrophoresis and Elutip-purified. The fragment was then 32P-labeled by random hexamer nucleotide priming (Amersham, UK) and purified by Sephadex G50 size exclusion chromatography (Pharmacia, Uppsala, Sweden). The blot was hybridized at 6x SSPS, 50% formamide at 48°C for 16 h and washed twice in 0.2x SSC, 0.05% sodium pyrophosphate, 1% SDS at 70°C and once in 0.2x SSC at room temperature. The filter was exposed for 20 h using Reflection film and intensifying screen (NEN Dupont). The autoradiographs were quantified using a laser scanning densitometer equipped with Image Quant software (Molecular Dynamics, Sunnyvale, CA). The results were corrected for differences in the amount of total RNA after hybridization to actin.
Determination of 3'-phosphoadenylsulfate: galactosylceramide 3'sulfotransferase mRNA in isolated rat islet cells
Total brain RNA from adult and 10- and 27-week-old rats and total RNA from rat islets were isolated and blotted as described above. A 3'-phosphoadenylsulfate:galactosylceranide 3'sulfotransferase probe was made using two internal PCR primers to amplify a 666 bp fragment from 1 ng pSV-hCST (Honke et al., 1997). The labeling reaction was performed in 25 µl using the buffer provided with the Taq polymerase: dATP, dGTP, and dTTp (5 µM each), 2.5µM 32P-dCTP (800 µCi/µl, NEN, DuPont), primers 0.2 µM each, 1 U Taq polymerase (ICN Pharmaceuticals, Costa Mesa, CA). Purification and hybridization were performed as described above. The filters were hybridized to a GAPDH probe to correct for differences in total RNA amounts.
Electron microscopy procedures
Islets of Langerhans were isolated from Lewis rats as described above. The tissues were fixed for 1 h in a mixture of 2.5% paraformaldehyde and 0.1% glutaraldehyde. After washings in cacodylate buffer pH 7.3 and dehydration in 70% alcohol, specimens were embedded in LR-white (Bio-Rad, Watford, UK). To avoid unspecific staining, the ultrasections were blocked by 0.1% bovine serum albumin and 0.05% Tween 20 (Sigma, St. Louis, MO) in TrisHCl, pH 8.2. Sections were then incubated with Sulf I, diluted 1:75 in the same buffer at +4°C for 24 h. After incubation for 1 h at +20°C with rabbit anti-mouse immunoglobulin (F261, Dako) diluted 1:100 and absorbed with rat serum, sections were washed and treated for 1 h at +20°C with goat anti-rabbit immunoglobulin conjugated with colloidal-gold (10 nm, Bio Cell Research Laboratories, Cardiff, UK) diluted 1:100. After washing, specimens were postfixed in 2% glutaraldehyde for 5 min and stained with uranyl acetate/lead citrate before examination in a Philips EM208 electron microscope. Controls were treated similarly, except for incubation with the primary antibody, and showed no staining.
The immunogold labeling of the Golgi apparatus was quantified in four experiments each with incubation of the islets in three different glucose concentrations (2.8, 11.0, and 20.0 mM). Ultramicrographs (5456) at a magnification of 30,000, showing an area of 5100 x 6900 nm of ß cells, were taken from each of the incubations. Labeling (noted as present or absent) was registered for the Golgi profile on every micrograph.
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