HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Buschard, K. Articles by Gromada, J. Search for Related Content PubMed PubMed Citation Articles by Buschard, K. Articles by Gromada, J. Social Bookmarking                What's this?

Sulfatide Controls Insulin Secretion by Modulation of ATP-sensitive K⁺-Channel Activity and Ca²⁺-Dependent Exocytosis in Rat Pancreatic ß-Cells

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
The glycosphingolipid sulfatide is present in secretory granules and at the surface of pancreatic ß-cells, and antisulfatide antibodies (ASA; IgG1) are found in serum from the majority of patients with newly diagnosed type 1 diabetes. Here we demonstrate that sulfatide produced a glucose- and concentration-dependent inhibition of insulin release from isolated rat pancreatic islets. This inhibition of insulin secretion was due to activation of ATP-sensitive K⁺-(K_ATP) channels in single rat ß-cells. No effect of sulfatide was observed on whole-cell Ca²⁺-channel activity or glucose-induced elevation of cytoplasmic Ca²⁺ concentration. It is interesting that sulfatide stimulated Ca²⁺-dependent exocytosis determined by capacitance measurements and depolarized-induced insulin secretion from islets exposed to diazoxide and high external KCl. The monoclonal sulfatide antibody Sulph I as well as ASA-positive serum reduced glucose-induced insulin secretion by inhibition of Ca²⁺-dependent exocytosis. Our data suggest that sulfatide is important for the control of glucose-induced insulin secretion and that both an increase and a decrease in the sulfatide content have an impact on the secretory capacity of the individual ß-cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

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 (K_ATP-channels) and stimulation of Ca²⁺-dependent exocytosis, whereas incubation of ß-cells with antisulfatide antibodies does not affect K_ATP-channel activity but decreases insulin secretion by reducing the rate of Ca²⁺-induced exocytosis.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Preparation of islets and single ß-cells.
Male Lewis rats (250–300 g) were purchased from Møllegaard (Ll; Skensved, Denmark). The rats were anesthetized by pentobarbital (100 mg/kg i.p.), and the pancreases were removed quickly. Pancreatic islets were then isolated by collagenase digestion and dispersed into single cells using dispase (Dispase II; Boehringer Mannheim, Mannheim, Germany), and the ß-cells were separated by fluorescence-activated cell sorting (8) using a FACSstar (Becton-Dickinson, Mountain View, CA). Window sortings were set to enclose the ß-cell population, which in the combined high-scatter and high-fluorescence fraction includes 97 ± 3% ß-cells (8).

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 2–4 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 (1.5–2 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 >35–40 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 17–38). 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 23–46). 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 42–75), 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 Student’s t test for unpaired observations.

	RESULTS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Effects of sulfatide on glucose-induced insulin secretion.
Figure 1A shows the effects of sulfatide (20 nmol/ml for 24 h) on insulin secretion from rat islets at increasing glucose concentrations in the extracellular medium. Sulfatide inhibited insulin release at 11.2 mmol/l glucose by 40% (P < 0.01). Short-term incubation with sulfatide (20 nmol/ml for 1 h) did not affect insulin release (data not shown). Figure 1B suggests that the inhibitory effect of sulfatide on insulin secretion is dependent on the dose. The islets were incubated for 24 h in the presence of 16.8 mmol/l glucose. No significant effect was observed at 5 and 10 nmol/ml, whereas maximal inhibition occurred at 20 nmol/ml sulfatide. Sulfatide did not affect insulin release when Ca²⁺ was depleted from the extracellular medium (control 2.6 ± 0.5 vs. 2.8 ± 0.9 ng · islet^-1 · h^-1 in the presence of sulfatide; data not shown). Finally, insulin secretion was not affected by the related glycolipids gal-cer and GM1. Under these conditions, insulin secretion amounted to 16.6 ± 1.5 (control), 16.2 ± 2.1 (gal-cer), and 16.8 ± 1.9 ng · islet^-1 · h^-1 (GM1).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Sulfatide inhibits glucose-induced insulin secretion in rat islets. A: Insulin release was measured for 24 h in groups of 10 size-matched islets at the indicated glucose concentrations in the absence (control; ) or presence of sulfatide (20 nmol/ml; •). B: Insulin secretion evoked by 16.8 mmol/l glucose was dependent on the concentration of sulfatide in the extracellular medium. The islets were exposed to sulfatide for 24 h. Data are mean ± SE of three different experiments, and each experiment represents the average of four samples. *P < 0.05; **P < 0.01.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Effects of sulfatide on whole-cell KATP-channel activity in single rat ß-cells. A: Whole-cell KATP currents were measured in response to 10 mV of de- and repolarizing voltage pulses from a holding potential of -70 mV using the standard whole-cell configuration and a pipette solution with 0.3 mmol/l ATP and 0.3 mmol/l ADP to activate the channels. The current traces reflect the maximal current recorded after full replacement of the cytoplasm with the pipette solution under control conditions and after pretreatment with sulfatide (20 nmol/ml for 30 min). B: Time course of increase in whole-cell current in cells exposed to sulfatide (20 nmol/ml; •) for the indicated periods of time. No change in the currents over time was observed in control cells (). Data are means ± SE of five to six different cells. *P < 0.05; **P < 0.01.

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).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Effects of sulfatide on whole-cell Ca2+-channel activity in rat ß-cells. Whole-cell Ca2+ currents were measured at the indicated membrane voltages and elicited from a holding potential of -70 mV using the standard whole-cell configuration. The currents were recorded under control conditions () and after pretreatment of ß-cells with sulfatide (20 nmol/ml; •) for 30 min. Data are means ± SE of five to seven different cells.

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 2–4 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).

View larger version (26K): [in this window] [in a new window]   FIG. 4. Sulfatide stimulates exocytosis in rat ß-cells. A: Increases in cell capacitance elicited by intracellular infusion of a pipette solution with a free Ca2+ concentration of 220 nmol/l under control conditions and in a cell incubated for 30 min with 20 nmol/ml sulfatide before start of the experiment. The traces depict changes in cell capacitance measured over the first 2 min after establishment of the whole-cell configuration. Throughout the recordings, the cells were clamped at -70 mV to avoid activation of the voltage-dependent Ca2+ channels that would otherwise interfere with the measurements. B: Average rates of exocytosis, measured 10–60 s after break-in into the cells, in cells incubated for 30 min in the absence () or in the presence () of 20 nmol/ml sulfatide using pipette-filling solutions with [Ca2+]i of 10, 220, and 870 nmol/l. Data are means ± SE of five or six different cells. *P < 0.05; **P < 0.01. C: Effects of sulfatide on insulin secretion in islets exposed to 250 µmol/l diazoxide, 25 mmol/l KCl, and 16.8 mmol/l glucose. Insulin secretion was measured for 24 h in groups of 10 size-matched islets in the absence () or presence () of 20 nmol/ml sulfatide. Data are means ± SE of three different experiments, and each experiment represents the average of four samples. *P < 0.05.

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).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Sulph I and ASA-positive diabetes serum inhibit glucose-induced insulin secretion in rat islets. A: Insulin release was measured for 24 h in groups of 10 size-matched islets at 16.8 mmol/l glucose and increasing concentrations of Sulph I in the extracellular medium. B: Insulin secretion evoked by 16.8 mmol/l glucose was inhibited by ASA-positive diabetes sera (ASA pos; five different patients) but not by ASA-negative sera from five different patients with newly diagnosed type 1 diabetes (ASA neg) or by five different control sera from healthy individuals. Sera were diluted 1:10, and insulin secretion was measured for 24 h. Data are means ± SE of three different experiments, and each experiment represents the average of four samples. *P < 0.05; **P < 0.01.

Sulph I and ASA-positive serum do not affect K_ATP-channel activity.
Figure 6A depicts recordings of whole-cell K_ATP 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 K_ATP conductance (Fig. 6B). A similar lack of effect on K_ATP-channel activity was observed in cells pretreated for only 30 min with Sulph I (Fig. 6B).

View larger version (23K): [in this window] [in a new window]   FIG. 6. Effects of Sulph I and ASA-positive serum on whole-cell KATP-channel activity in rat ß-cells. A: Whole-cell KATP currents were measured in response to 10 mV of de- and repolarizing voltage pulses from a holding potential of -70 mV using the standard whole-cell configuration (as described in the legend to Fig. 2) under control conditions (control) and after pretreatment with Sulph I (20 µg/ml) or ASA-positive serum (dilution 1:10) for 24 h. B: Average increase in the whole-cell current in control cells () and in cells exposed to Sulph I (20 µg/ml) for 30 min or 24 h and to ASA-positive serum for 24 h (dilution 1:10; ). Data are means ± SE of five to eight different cells.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Sulph I and ASA-positive diabetes serum reduce Ca2+-evoked exocytosis in rat ß-cells. A: Increases in cell capacitance elicited by intracellular infusion with a Ca2+-EGTA buffer with a free Ca2+ concentration of 870 nmol/l in the absence (control) and presence of Sulph I (cells incubated with 20 µg/ml for 30 min before initiation of the experiment). B: Average rates of increase in cell capacitance measured 10–60 s after establishment of the whole-cell configuration in cells incubated for 30 min in the absence () or in the presence () of 20 µg/ml Sulph I using pipette-filling solutions with [Ca2+]i of 10, 220, and 870 nmol/l. C: Increases in cell capacitance measured over the first 2 min after establishment of the whole-cell configuration in cells pretreated with control serum (P1), ASA-positive type 1 diabetes serum (P6), or ASA-negative type 1 diabetes serum (P11; all diluted 1:10) for 30 min before initiation of the experiments. D: Average rates of increase in cell capacitance measured 10–60 s after establishment of the whole-cell configuration in cells preincubated for 30 min with five different control sera (control), five different ASA-positive type 1 diabetes sera (ASA pos), or five different ASA-negative type 1 diabetes sera (ASA neg). E: Average rates of increase in cell capacitance (10–60 s) in cells exposed to ASA-positive serum before (P8), after its passage through a protein A column to remove IgG (P8 pel), and to the ASA-protein A IgG–absorbed fraction (P8 prot A). F: Average rates of increase in cell capacitance (10–60 s) before (+ASA; ) and after (-ASA; ) removal of ASA by passage of three different ASA-positive type 1 diabetes sera over a sulfatide-coated Sephadex column. Data are means ± SE of five or six different cells. *P < 0.05, **P < 0.01.

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 IgG–absorbed 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 4–6 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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
The rate of insulin secretion is dependent on the external glucose concentration and is secondary to its uptake and metabolism by the ß-cell. The resulting increase in the ATP-to-ADP ratio leads to closure of the K_ATP channels, membrane depolarization, and activation of the voltage-dependent Ca²⁺ channels. The ensuing rise in the [Ca²⁺]_i stimulates, through a series of less well-defined steps, exocytosis of the insulin-containing granules. Here we demonstrate that sulfatide plays an important role in controlling glucose-induced insulin secretion and that the glycolipid interacts with both proximal and distal regulatory steps in the ß-cell stimulus-secretion coupling.

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 membrane–associated 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 K_ATP 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 K_ATP channels in the ß-cell plasma membrane (22,23), and it is tempting to speculate that sulfatide stimulated K_ATP-channel activity by a related mechanism. It is interesting that ASA (Sulph I or ASA-positive serum) did not affect K_ATP-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 K_ATP 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 K_ATP 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 K_ATP-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 K_ATP 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.

	ACKNOWLEDGMENTS

 
The study was supported in part by a grant from Swedish Medical Research Council (K2000-03X-09909-09A).

We thank chief physician Henrik Permin for providing the Guillain-Barré sera and Jens Peter Stenvang for excellent technical assistance.

	FOOTNOTES

 
Address correspondence and reprint requests to Karsten Buschard, MD, Bartholin Instituttet, Kommunehospitalet, DK-1399 Copenhagen K, Denmark. E-mail: buschard{at}dadlnet.dk.

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; K_ATP channel, ATP-sensitive K⁺ channel; gal-cer, galactosylceramide.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Buschard K, Fredman P: Sulphatide as an antigen in diabetes mellitus. Diabetes Nutr Metab4 :221 –228,1996
2. Buschard K, Josefsen K, Hansen SV, Horn T, Marshall MO, Persson H, Månsson J-E, Fredman P: Sulphatide in islets of Langerhans and in organs affected in diabetic late complications: a study in human and animal tissue. Diabetologia37 :1000 –1006,1994[Medline]
3. Bovin LF, Fredman P, Månsson J-E, Buschard K, Bendtzen K: In vitro production of cytokines is influenced by sulfatide and its precursor galactosylceramide. FEBS Lett455 :339 –343,1999[Medline]
4. Vos JP, Lopes-Cardozo M, Gadella BM: Metabolic and functional aspects of sulphogalactolipids. Biochim Biophys Acta1211 :125 –149,1994[Medline]
5. Yamaguchi S, Miyazaki Y, Oka S, Yano I: Stimulation of phagocytosis and phagosome-lysosome (P-L) fusion of human polymorphonuclear leukocytes by sulfatide (galactosylceramide-3-sulfate). FEMS Immunol Med Microbiol13 :107 –111,1996[Medline]
6. Buschard K, Schloot NC, Kaas A, Bock T, Horn T, Fredman P, Roep BO: Immunomodulation of insulin-specific autoreactive T-cells by sulfatide which is variably expressed in beta cells. Diabetologia42 :1212 –1218,1999[Medline]
7. Buschard K, Josefsen K, Horn T, Fredman P: Sulphatide and sulphatide antibodies in insulin-dependent diabetes mellitus. Lancet342 :840 ,1993[Medline]
8. Josefsen K, Stenvang JP, Kindmark H, Berggren PO, Horn T, Kjær T, Buschard K: Fluorescence-activated cell sorted rat islet cells and studies of the insulin secretory process. J Endocrinol149 :145 –154,1996[Abstract/Free Full Text]
9. Martell AE, Smith RM: Critical Stability Constants. Vols. 1 and 2. New York, Plenum Press,1971
10. Månsson J-E, Mo H, Egge H, Svennerholm L: Trisialosyllactosylceramide (GT3) is ganglioside in human lung. FEBS Lett196 :259 –262,1986[Medline]
11. Månsson J-E, Fredman P, Bigner DD, Molin K, Rosengren B, Friedman HS, Svennerholm L: Characterization of new gangliosides of the lactotetraose series in murine xenografts of a human glioma cell line. FEBS Lett201 :109 –113,1986[Medline]
12. Fredman P, Månsson J-E, Rynmark B-M, Josefsen K, Ekblond A, Halldner L, Osterbye T, Horn T, Buschard K: The glycosphingolipid sulfatide in the islets of Langerhans in rat pancreas is processed through recycling: possible involvement in insulin trafficking. Glycobiology10 :39 –50,2000[Abstract/Free Full Text]
13. Fredman P, Mattsson L, Andersson K, Davidsson P, Ishizuka I, Jeansson S, Månsson J-E, Svennerholm L: Characterization of the binding epitope of a monoclonal antibody to sulphatide. Biochem J251 :17 –22,1988[Medline]
14. Bansal R, Warrington AE, Garel AL, Pfeiffer SE: Multiple and novel specificities of monoclonal antibodies O1, O4, R-mAb used in the analysis of oligodendrocyte development. J Neurosci Res24 :548 –557,1989[Medline]
15. Bokvist K, Eliasson L, Ämmälä C, Renström E, Rorsman P: Co-localization of L-type Ca²⁺ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis. EMBO J14 :50 –57,1995[Medline]
16. Smith PA, Ashcroft FM, Rorsman P: Simultaneous recordings of glucose dependent electrical activity and ATP-regulated K⁺-currents in isolated mouse pancreatic ß-cells. FEBS Lett261 :187 –190,1990[Medline]
17. Trube G, Rorsman P, Ohno-Shosaku T: Opposite effects of tolbutamide and diazoxide on the ATP-dependent K⁺-channel in mouse pancreatic ß-cells. Pflügers Arch407 :493 –499,1986[Medline]
18. Gembal M, Gilon P, Henquin JC: Evidence that glucose can control insulin release independently from its action on ATP-sensitive K⁺ channels in mouse B-cells. J Clin Invest89 :1288 –1295,1992[Medline]
19. Fredman P, Vedeler CA, Nyland H, Aarli JA, Svennerholm L: Antibodies in sera from patients with inflammatory demyelinating polyradiculoneuropathy react with ganglioside LM1 and sulphatide of peripheral nerve myelin. J Neurol238 :75 –79,1991[Medline]
20. Holmgren J, Lönnroth I, Månsson J-E, Svennerholm L: Interaction of cholera toxin and membrane GM1 ganglioside of small intestine. Proc Natl Acad Sci U S A72 :2520 –2524,1975[Abstract/Free Full Text]
21. Cuatrecasas P: Gangliosides and membrane receptors for cholera toxin. Biochemistry12 :3558 –3566,1973[Medline]
22. Bränström R, Corkey BE, Berggren P-O, Larsson O: Evidence for a unique long chain acyl-CoA ester binding site on the ATP-regulated potassium channel in mouse pancreatic beta cells. J Biol Chem272 :17390 –17394,1997[Abstract/Free Full Text]
23. Gribble FM, Proks P, Corkey BE, Ashcroft FM: Mechanism of cloned ATP-sensitive potassium channel activation by oleoyl-CoA. J Biol Chem273 :26383 –26387,1998[Abstract/Free Full Text]
24. Eliasson L, Renström E, Ämmälä C, Berggren P-O, Bertorello AM, Bokvist K, Chibalin A, Deeney JT, Flatt PR, Gäbel J, et al.: PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic ß cells. Science271 :813 –815,1996[Abstract]
25. Deeney JT, Gromada J, Høy M, Olsen HL, Rhodes CJ, Prentki M, Berggren P-O, Corkey BE: Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secretion cells (HIT T-15 and NMRI ß-cells). J Biol Chem275 :9363 –9368,2000[Abstract/Free Full Text]
26. Juntti-Berggren L, Larsson O, Rorsman P, Ämmälä C, Bokvist K, Wåhlander K, Nicotera P, Dypbukt J, Orrenius S, Hallberg A, et al: Increased activity of L-type Ca²⁺ channels exposed to serum from patients with type 1 diabetes. Science261 :86 –90,1993[Abstract/Free Full Text]
27. Rabinowe SL: Immunology of diabetic and polyglandular neuropathy. Diabetes Metab Rev6 :169 –188,1990[Medline]
28. Hsu FF, Bohrer A, Turk J: Electrospray ionization tandem mass spectrometric analysis of sulfatide: determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets. Biochim Biophys Acta1392 :202 –216,1998[Medline]

CiteULike

Del.icio.us

Digg

Reddit

Technorati

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Buschard, K. Articles by Gromada, J. Search for Related Content PubMed PubMed Citation Articles by Buschard, K. Articles by Gromada, J. Social Bookmarking                What's this?