1 Bartholin Instituttet, Kommunehospitalet, Copenhagen, Denmark
2 Laboratory of Islet Cell Physiology, Novo Nordisk, Bagsværd, Denmark
3 Department of Endocrinology, Hvidovre Hospital, Hvidovre, Denmark
4 Department of Clinical Neuroscience, Section of Neurochemistry, University of Göteborg, Mölndals Hospital, Mölndal, Sweden
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ABSTRACT |
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INTRODUCTION |
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Sulfatide comprises both a hydrophilic and a hydrophobic part and by the sulfate group a negative charge. Although not fully elucidated, several biological functions have been assigned to sulfatide, including 1) altered cytokine production from mononuclear cells (3), 2) facilitation of ionic transportation in kidney tubule cells (4), and 3) fusion of phagosomes and lysosomes (5). Furthermore, the amount of sulfatide in the secretory granules in ß-cells exposed to high glucose levels seems to be reduced, indicating that under certain circumstances sulfatide synthesis may be insufficient (6).
Antisulfatide antibodies (ASA) have been found in the majority of serum samples from patients with newly diagnosed type 1 diabetes in titers up to 1:3,200 (7). The antibodies were of the IgG1 class and have recently also been found in prediabetic individuals (P.F. and K.Bu., unpublished observations) but not in serum from healthy control subjects.
The aim of this study was to explore the possible influence on insulin secretion by sulfatide, by type 1 diabetes serum with ASA, and by a specific antisulfatide monoclonal antibody, Sulph I. To this end, we applied patch clamp techniques and capacitance measurements of exocytosis to rat ß-cells. We demonstrate here that sulfatide is required for normal insulin secretion and that an increase in the ß-cell sulfatide content leads to activation of ATP-sensitive K+ channels (KATP-channels) and stimulation of Ca2+-dependent exocytosis, whereas incubation of ß-cells with antisulfatide antibodies does not affect KATP-channel activity but decreases insulin secretion by reducing the rate of Ca2+-induced exocytosis.
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RESEARCH DESIGN AND METHODS |
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Electrophysiology.
Patch pipettes were pulled from borosilicate glass capillaries, coated with Sylgard near their tips and fire-polished before use. The pipette resistance (when filled with the pipette-filling solutions) was 24 M. The zero-current potential was adjusted before establishment of the seal with the pipette in the bath. Whole-cell K+ currents were estimated by applying 10 mV hyper- and depolarizing voltage pulses (duration 200 ms; pulse interval 2 s) from a holding potential of -70 mV. The currents were recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA), digitized, and stored in a computer using the Digidata AD-converter (Axon Instruments) and the software pClamp (Version 6.0; Axon Instruments). Whole-cell Ca2+ currents were evoked by 100-ms membrane depolarization (range -60 to 60 mV in 10-mV increments) from a holding potential of -70 mV using an EPC-9 patch clamp amplifier and the Pulse software (Version 8.31; HEKA Elektronik, Lamprecht/Pfalz, Germany). Exocytosis was monitored in single ß-cells as increases in cell membrane capacitance (sample rate 2.5 Hz) using the EPC-9 amplifier and the Pulse software. The standard whole-cell configuration was used throughout this study, except when mentioned otherwise.
Solutions.
The extracellular medium consisted of (in mmol/l) 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.4 with NaOH), and 5 D-glucose. The extracellular solution used for recordings of whole-cell Ca2+ currents were (in mmol/l) 118 NaCl, 20 tetraethylammonium, 5.6 KCl, 10 CaCl2, 5 HEPES, and 5 glucose. The volume of the recording chamber was 0.4 ml, and the solution entering the bath (1.52 ml/min) was maintained at 33°C. For measurements of standard whole-cell K+-channel activity, the pipette solution contained (in mmol/l) 125 KCl, 30 KOH, 10 EGTA, 1.3 MgCl2, 5 HEPES, 0.3 Mg-ATP, and 0.3 K-ADP (pH 7.15). For recordings of perforated-patch whole-cell K+-channel activity, the following pipette-filling solution was used (in mmol/l): 76 K2SO4, 10 NaCl, 10 KCl, 1 MgCl2, and 5 HEPES (pH 7.35, KOH). Electrical contact was established by adding 0.24 mg/ml amphotericin B to the pipette solution. Perforation required a few minutes; the voltage clamp was considered satisfactory when the series conductance was constant and >3540 ns. For recordings of whole-cell Ca2+ currents, the pipette solution was composed of (in mmol/l) 125 Cs-glutamate, 1 MgCl2, 2 CaCl2, 10 EGTA, 5 HEPES, and 3 Mg-ATP (pH 7.15 with CsOH). Exocytosis was elicited by infusion of the following Ca2+/EGTA buffers through the recording pipette (in mmol/l): 125 K-glutamate; 10 KCl; 10 NaCl; 1 MgCl2; 5 HEPES; 3 Mg-ATP; 10 EGTA; and 0, 5, or 8 CaCl2. The [Ca2+]i of the resulting buffers were 10, 220, or 870 nmol/l using the binding constants of Martell and Smith (9).
Glycolipids.
The following glycolipids were used: sulfatide (3'sulfogalactosylceramide), gal-cer, and GM1. All three molecules are composed of a hydrophilic sugar chain and a hydrophobic part consisting of ceramide, i.e., sphingosine and fatty acid. Gal-cer is the precursor of sulfatide and lacks the negatively charged sulfate group. The GM1 has a negative charge like sulfatide but in this case expressed by sialic acid. All of the glycolipids were isolated from porcine brain, and their structures were analyzed by fast atom bombardment mass spectrometry (10,11). It is important to emphasize that the composition of sulfatide used in this study consists of molecules, which differ in length and degree of saturation of the fatty acid chain (12). Stock solutions of each glycolipid were made in chloroform/methanol/water (60:30:4.5 vol/vol/vol) in concentrations of 1 µmol/ml. The tubes were sealed and after 1 h at room temperature, stored at 4°C. The glycolipids were transferred to glass tubes before use and evaporated at room temperature overnight. The dried glycolipids were redissolved in phosphate-buffered saline (pH 7.1) and sonicated for 20 s using a Branson sonicator (Danbury, CT). The glycolipids were used within 2 h.
Monoclonal antibodies.
The antisulfatide monoclonal antibody Sulph I was raised after immunizing BALB/c mice with sulfatide coated on Salmonella minnesota bacterial membrane (13). The subclass of the antibody is IgG1, and it reacts specifically with sulfatide and the related structures lactosylceramide sulfate and seminolipid, of which only sulfatide is present in adult islets (2). For controls, the following two monoclonal antibodies (both IgG1) were used: antigalactosylceramide (14) and antiglucose oxidase (Dako, Copenhagen, Denmark).
Patient serum.
Sera from 11 patients with newly developed type 1 diabetes were taken within 1 week of diagnosis. The patients were four women and seven men with a mean age of 27 ± 7 years (range 1738). All fulfilled the following criteria at time of diagnosis: random blood glucose concentration >12 mmol/l and significant ketonuria and glucosuria but not overweight. Insulin treatment was initiated on the day of diagnosis. The study was performed in accordance with the principles of the Declaration of Helsinki. Five of the patients displayed ASA in titers between 1:800 and 1:1,600 as determined by thin-layer chromatography (7). Six patients were ASA-negative. As additional control, ASAs were removed from three ASA-positive patient sera by passing sera six times through an octylsepharose column coated with sulfatide. In addition, ASA-positive patient serum was run twice through a protein A column (Pharmacia, Uppsala, Sweden) and the samples with (eluate) or without IgG were used for capacitance measurements. The control group consisted of three women and two men with an average age of 28 ± 10 years (range 2346). All subjects were healthy with no known allergies and with no family history of type 1 diabetes. Furthermore, sera from five patients (three women and two men) with Guillain-Barré syndrome were examined. These patients had no history of type 1 diabetes. Their average age was 57 ± 7 years (range 4275), and they displayed ASA titers between 1:800 and 1:3,200.
Measurements of [Ca2+]i.
The [Ca2+]i measurements were made using an Axiovert 135 inverted microscope equipped with a Plan-Neofluar 100x/1.30 objective (Carl Zeiss, Oberkochen, Germany) and an Ionoptix fluorescence imaging system (Milton, MA) as described elsewhere (15). Before the experiments, the cells were loaded with 0.2 µmol/l fura-2/AM (Molecular Probes, Eugene, OR) for 20 min.
Insulin secretion experiments.
Insulin release was measured at 37°C after static incubation. Groups of 10 size-matched islets isolated from Lewis rats were cultured overnight in RPMI 1640 medium and preincubated for 20 min in modified RPMI-1640 medium supplemented with 1% penicillin/streptomycin and 3 mmol/l glucose in 96-well Durapore membrane plates (Millipore, Molsheim, France). The preincubation medium was aspirated using a vacuum control pump (Millipore) and discarded. The islets were then resuspended in 200 µl of RPMI-1640 with different glucose concentrations and in the absence or presence of test compounds. At the end of the test incubation, the medium was aspirated and kept at -20°C until assayed for insulin using the enzyme-linked immunosorbent assay technique. Each data point represents the average of three individual experiments from different islet preparations, and each experiment was performed in quadruplicate.
Data analysis.
The effects of test substances on insulin release are expressed in percentage of control. The exocytotic rate is presented as the increase in cell capacitance occurring during the first 60 s after establishment of the whole-cell configuration excluding any rapid changes occurring during the initial 10 s required for equilibration of the pipette solution with cytosol. All voltage signals were filtered at 500 Hz and sampled at a rate of 1 kHz. Results are presented as mean ± SE for the indicated number of experiments. Statistical significance was evaluated using Students t test for unpaired observations.
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RESULTS |
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Measurements of Ca2+-channel activity and [Ca2+]i.
Incubation of ß-cells with sulfatide (20 nmol/ml for 30 min) did not affect the activity of the voltage-gated Ca2+ currents recorded during 100-ms voltage-clamp depolarization from -70 mV to membrane potentials between -60 and 60 mV with 10-mV increments (Fig. 3). Similar results were observed after incubation of the cells with 20 µg/ml sulfatide for 24 h (data not shown).
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Sulfatide stimulates exocytosis.
Figure 4A shows that infusion of a single rat ß-cell with a Ca2+/EGTA buffer with a free Ca2+ concentration of 220 nmol/l produced a small increase in cell capacitance (indicative of exocytosis) after establishment of the whole-cell configuration. In general, the rate of exocytosis reached a new steady-state level within 24 min. It is evident that preincubation of cells with sulfatide (20 nmol/ml for 30 min) exerted a strong stimulation (4.5-fold) of exocytosis (Fig. 4A). On average (Fig. 4B), exocytosis increased from 2.0 ± 0.6 (n = 5) to 8.7 ± 0.8 fF/s (n = 6; P < 0.01) in cells that were pretreated with sulfatide. Similar results were observed after incubation of cells for 1 h with sulfatide (4.2-fold stimulation) and after inclusion of sulfatide in the pipette-filling solution (4.3-fold enhancement). No stimulation of exocytosis was observed with gal-cer (2.2 ± 0.5 fF/s; n = 5) or GM1 (1.9 ± 0.6 fF/s; n = 5; cells incubated with 20 nmol/ml for 30 min; data not shown).
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Monoclonal sulfatide antibody Sulph I and diabetes ASA-positive serum reduce glucose-induced insulin secretion.
In the presence of 16.8 mmol/l glucose, the monoclonal sulfatide antibody Sulph I produced a dose-dependent inhibition of insulin secretion from rat islets (Fig. 5A). No significant inhibition was observed at 5 or 10 µg/ml, whereas 20 µg/ml Sulph I produced a maximal inhibition of insulin release by 42% (P < 0.01). Sulph I did not affect insulin release when Ca2+ was depleted from the extracellular medium (control 2.2 ± 0.7 vs. 2.6 ± 0.8 ng · islet-1 · h-1 in the presence of Sulph I; data not shown). Furthermore, insulin secretion was not affected by Sulph I boiled for 30 min or the related monoclonal antibodies against gal-cer or glucose oxidase (both 20 µg/ml; data not shown).
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Sulph I and ASA-positive serum do not affect KATP-channel activity.
Figure 6A depicts recordings of whole-cell KATP currents under control conditions and in cells exposed to either 20 µg/ml Sulph I or ASA-positive serum (diluted 1:10) for 24 h. The treatment did not affect the magnitude of the whole-cell KATP conductance (Fig. 6B). A similar lack of effect on KATP-channel activity was observed in cells pretreated for only 30 min with Sulph I (Fig. 6B).
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Figure 7E shows that when ASA-positive serum (P8) was passed through a protein A column to remove the IgG fraction (P8 pel; titer <1:100), no inhibition of exocytosis was observed. By contrast, the ASA protein A IgGabsorbed fraction (P8 prot A fraction; titer 1:800) decreased the rate of exocytosis by 35% (P < 0.01; n = 5; Fig. 7E). Furthermore, removal of ASA by passage over a sulfatide-coated Sephadex column (three different ASA-positive sera) abolished the inhibitory effect on exocytosis (Fig. 7F; -ASA). Under these experimental conditions, the average rate of increase in exocytosis (8.7 ± 0.5 fF/s; n = 26) was not different from that of cells treated with control sera (8.4 ± 0.4 fF/s; n = 25; Fig. 7D). For clarity, the corresponding increases in exocytosis for ASA-positive sera are depicted in Fig. 7F (+ASA) and amounted to 3.4 ± 0.4 fF/s (P < 0.01; n = 26).
ASA-positive sera from patients with Guillain-Barré syndrome do not affect insulin secretion and exocytosis.
Patients with the neurological disease Guillain-Barré syndrome (inflammatory demyelinating polyradiculoneuropathy) display antibodies against sulfatide, which is present in the myelin sheets around the peripheral nerves (19). It is interesting that exposure of rat ß-cells for 30 min to five different ASA-positive sera from patients with Guillain-Barré syndrome (diluted 1:10) did not affect the average rates of increase in exocytosis (rates between 8.4 and 8.8 fF/s compared with 8.0 fF/s under control conditions). Correspondingly, increasing the exposure time to 46 h did not affect exocytosis (8.9 ± 0.4 vs. 8.8 ± 0.3 fF/s for control; n = 5). In parallel experiments, however, exposure of cells to ASA-positive type 1 diabetes sera decreased the rate of exocytosis by 59% (data not shown). In agreement with these data, glucose-induced insulin secretion (16.8 mmol/l) from rat islets was not affected by a 24-h treatment with ASA-positive sera from patients with Guillain-Barré syndrome: control serum 15.1 ± 1.1 ng · islet-1 · h-1 and ASA-positive serum 15.6 ± 0.9 ng · islet-1 · h-1 (data not shown). Again, ASA-positive serum inhibited insulin secretion evoked by 16.8 mmol/l glucose by 37% (P < 0.05).
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DISCUSSION |
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It is known that exogenously added glycosphingolipids are inserted into the plasma membrane (20,21). It is reasonable to assume, therefore, that an increase in the plasma membrane concentration of sulfatide is responsible for the present observations. The one control glycolipid used, GM1, will also be incorporated into the plasma membrane. It is more water-soluble than sulfatide but does form micelles. The other control molecule, gal-cer, has a low solubility, so much less is taken up by the membrane and micelles are more stable.
The turnover rate of plasma membraneassociated glycosphingolipids is not known, and theoretically there is a possibility that exogenously added sulfatide, GM1, and gal-cer are endocytosed and degradation products produced in the lysosomes being responsible for the effects. However, this seems unlikely because they all give rise to the same final degradation products.
In the current study, we demonstrated that sulfatide produced a 40% increase in the whole-cell KATP conductance and caused inhibition of glucose-induced insulin secretion. It was demonstrated previously that acute addition of long-chain acyl-CoA esters, the active intracellular form of free fatty acids, activate KATP channels in the ß-cell plasma membrane (22,23), and it is tempting to speculate that sulfatide stimulated KATP-channel activity by a related mechanism. It is interesting that ASA (Sulph I or ASA-positive serum) did not affect KATP-channel activity. The reason for this is unknown, but we speculate that binding of the antibody to sulfatide in the plasma membrane does not affect the ability of sulfatide to interact with the channel protein. Alternatively, the membrane areas around the KATP channels, unlike the exocytose-fusing membrane areas, are not associated with appreciable amounts of sulfatide unless exogenous sulfatide is added.
Stimulation of insulin secretion by direct modulation of the secretory process distal to a rise in [Ca2+]i is physiologically important. For example, it was demonstrated previously that the hypoglycemic sulfonylureas accelerate Ca2+-dependent exocytosis in mouse ß-cells and that the direct effect of the sulfonylurea tolbutamide on exocytosis may account for up to 75% of the total stimulatory action (24). It is interestingly that the effect of sulfatide on exocytosis is of the same magnitude as that of tolbutamide. However, it is important to emphasize that these effects on exocytosis are secondary to closure of the KATP channels and the associated increase in [Ca2+]i. This is consistent with the lack of effect of sulfatide on exocytosis in the absence of intracellular Ca2+ and that stimulation of KATP-channel activity by sulfatide leads to inhibition of insulin secretion. We recently reported that acute stimulation with long-chain acyl-CoA enhances exocytosis in ß-cells (25). It is likewise tempting to speculate, in analogy to the effects on KATP channels, that sulfatide may stimulate exocytosis by a similar mechanism.
We demonstrate that both Sulph I and ASA-positive sera from subjects with newly diagnosed type 1 diabetes inhibit Ca2+-induced exocytosis. Evidence that ASA in ASA-positive sera mediate the inhibitory action on exocytosis comes from the observations that ASA-negative sera from patients with type 1 diabetes did not affect the rate of capacitance increase. Furthermore, ASA-positive sera passed through a protein A column was without inhibitory effect on exocytosis, whereas the ASA effluent from the column decreased the exocytotic capacity of the ß-cells to a similar extent as the corresponding ASA-positive sera. Passage over a sulfatide-coated octylsepharose column also removed the inhibitory activity. Finally, monoclonal antibodies of the IgG1 subtype against gal-cer and glucose oxidase did not affect exocytosis. These data support the observation that sulfatide is important for the fusion of secretory granules with the plasma membrane and suggest that the ASAs inhibit secretion by reducing the effective concentration of this lipid in the plasma membrane. This could result from an inability of sulfatide to participate in the exocytosis process upon binding to the antibody.
It was demonstrated previously that serum (IgM fraction) from patients with type 1 diabetes increase L-type Ca2+-channel activity in rat ß-cells (26). The subsequent increase in the [Ca2+]i was associated with apoptosis, and it was suggested that an IgM-mediated increase in Ca2+ influx might be part of the autoimmune reaction associated with type 1 diabetes (26). In contrast, treatment of ß-cells in the present study with either sulfatide or Sulph I was not associated with a significant change in the amplitude of the whole-cell Ca2+ current. Similar negative data were obtained after exposure of ß-cells to ASA-positive diabetes sera (data not shown).
The association of type 1 diabetes with Stiff-man syndrome and Guillain-Barré syndrome (27) suggests common antigens between the pancreatic ß-cells and neural tissue. One such antigen might be sulfatide, which is also a neural epitope to which 65% of sera from patients with Guillain-Barré syndrome react (19). However, incubation of ß-cells with ASA-positive sera from patients with Guillain-Barré syndrome did not modify the capacity of these cells to secrete. This suggests that the ASAs associated with the development of Guillain-Barré syndrome do not recognize the same epitope(s) as the ASA-positive sera from patients with type 1 diabetes. This may correspond well with the finding that the fatty acid composition of sulfatide in the brain is different from that of sulfatide in the islets, which has a high proportion of short fatty acid C16:0 and a lack of hydroxy fatty acids (12,28).
Collectively, our data are consistent with a role for sulfatide as a prime signal molecule that interacts with both proximal and distal regulatory steps in the ß-cell stimulus-secretion coupling. When released from the secretory granules, sulfatide may act as a physiological negative feedback mechanism on insulin secretion from the individual ß-cells or islets, but only when the glucose concentration is at a certain stimulatory level (see Fig. 1A). Our findings suggest that a fine balance between the amount of sulfatide in the ß-cell plasma membrane and the level of circulating ASAs may control insulin secretion. This observation may have implications for the understanding of the pathophysiology of type 1 diabetes because ASAs, which impair insulin secretion, have been found in the majority of serum samples from patients with newly diagnosed type 1 diabetes but also in prediabetic individuals. Depending on the time of occurrence of ASAs during the prediabetic phase, which is presently unknown, it is possible that ASAs may stress either the healthy ß-cells or ß-cells already affected by the insulitis process.
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ACKNOWLEDGMENTS |
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We thank chief physician Henrik Permin for providing the Guillain-Barré sera and Jens Peter Stenvang for excellent technical assistance.
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FOOTNOTES |
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Received for publication 14 February 2001 and accepted in revised form 25 April 2002.
The current affiliation of K.Bo. and J.G. is Lilly Research Laboratories, Hamburg, Germany.
ASA, antisulfatide antibodies; [Ca2+]i, free cytoplasmic Ca2+ concentration; KATP channel, ATP-sensitive K+ channel; gal-cer, galactosylceramide.
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REFERENCES |
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