Original Article
Inhibition of Glucose Transport into Rat Islet Cells by Immunoglobulins from Patients with New-Onset Insulin-Dependent Diabetes Mellitus
N Engl J Med 1990; 322:653-659March 8, 1990
- Abstract
- Abstract
Because glucose-stimulated insulin secretion is selectively impaired during the development of insulin-dependent diabetes mellitus (IDDM), we tested the possibility that the glucose transporter of pancreatic islet beta cells is a target of the autoimmune process in patients with IDDM.
We measured the uptake of 3-O-methyl-β-D-glucose by dispersed islet cells from rats after a 15-minute incubation with purified IgG from 27 patients with newly diagnosed IDDM, 28 normal subjects, and 5 patients with non-insulin-dependent diabetes mellitus (NIDDM). The IgG fractions from 26 of the 27 patients with IDDM (96 percent), but from none of the 5 patients with NIDDM, reduced the initial rates of 3-O-methyl-β-D-glucose uptake to at least 1 SD below the mean of the rates observed in the presence of IgG fractions from normal subjects (P<0.001 ). In contrast, the uptake of L-leucine by islet cells was not affected by any of the IgG fractions. The inhibitory activity of IgG from the patients with IDDM was abolished by preincubation with islet cells and membranes from hepatocytes, which contain the same glucose transporter as beta cells, but not with erythrocytes, which do not contain this transporter.
We conclude that IgG from patients with IDDM of recent onset, but not from those with NIDDM, inhibits glucose uptake by rat islet cells. The results are consistent with the presence of an antibody against a protein involved in glucose transport by beta cells that would thereby impair glucose-stimulated insulin secretion. (N Engl J Med 1990; 322:653–9.)
Article Activity
- Article
CLASSIC immunologic techniques have thus far failed to establish the identity of the beta-cell surface proteins that serve as the antigens for the islet-cell autoantibodies found in the serum of patients with insulin-dependent diabetes mellitus (IDDM). It is known that immunoglobulins from patients with new-onset IDDM block glucose-stimulated insulin secretion by perfused rat islet cells1 and that the acute insulin response to glucose but not to nonglucose secretagogues is attenuated in the interval before IDDM becomes overt in humans2 and rats.3 These facts have led us to consider that the target antigen might be a beta-cell surface protein involved in glucose recognition — for example, the glucose transporter. We therefore examined the effect of purified IgG from patients with new-onset IDDM on the initial rates of uptake of 3-O-methyl-β-D-glucose by dispersed islet cells from rats.
Methods
Study Populations
Serum for IgG purification was obtained from 27 patients with new-onset IDDM (6 to 34 years of age), 28 normal subjects (22 to 64 years of age), and 5 patients with non-insulin-dependent diabetes (NIDDM) (55 to 64 years of age). In 26 of the patients with IDDM, the serum was obtained within two months of diagnosis; in one patient the diagnosis had been made eight months earlier. Each serum sample was assigned a code number, and all subsequent work was performed on coded specimens. The IgG fractions from all diabetic patients were tested for the presence of cytoplasmic islet-cell antibodies according to the method of Krell and Rabin.4 All participants gave informed consent, and the study was approved by the institutional review board.
Purification of IgG
Protein A–Sepharose gel (Pharmacia Fine Chemicals, Uppsala, Sweden) was prepared by extensive washing and equilibration with 0.1 M sodium phosphate (pH 7.0). Each serum sample was diluted with an equal volume of 0.1 M sodium phosphate (pH 7.0), and 4 ml of the sample was mixed with 2 ml of protein A–Sepharose. After a 30-minute incubation at room temperature, the suspension was transferred to a column and washed with 20 ml of 0.1 M sodium phosphate (pH 7.0). IgG was eluted from the column with 10 ml of 0.5 M acetic acid in 0.89 percent saline (pH 3.0). After the elution of the IgG from the column, the pH of the eluate was immediately adjusted to 7.0 with 3.3 M TRIS (free base). The eluate was concentrated and dialyzed against phosphate-buffered saline. The mean (±SE) concentration of IgG was 9.0±0.3 mg per milliliter.
Isolation and Dispersal of Islets
Islets for each experiment were isolated from the pancreatic tissue of 15 to 20 male Wistar rats (weight, 200 to 250 g) according to a modification of the method of Naber et al.5 Briefly, the tissue was incubated with 20 ml of chilled Hanks' balanced salt solution (HBSS) containing 0.5 percent collagenase (pH 7.25) at 38.5°C for 16 minutes with occasional vigorous shaking. The digestion was stopped by adding chilled HBSS containing 0.5 percent bovine serum albumin, and the digested tissue was washed three times in HBSS containing 0.5 percent bovine serum albumin. The final sediment was resuspended in 4 ml of 25 percent Ficoll in HBSS and was overlaid with 2 ml each of 23 percent, 20.5 percent, and 11 percent Ficoll (wt/wt). After centrifugation at 700×g for 15 minutes at 4°C, the islets were harvested from the 11 percent—20.5 percent Ficoll interface with a plastic pipette. The Ficoll was removed by washing the islets with HBSS containing 0.5 percent bovine serum albumin and centrifuging at 700×g for two minutes at room temperature. Between 5000 and 8000 islets were routinely obtained from 20 rats.
The purified islets were resuspended in HBSS that was free of calcium and magnesium and contained 3 mM ethylene glycol bis(βaminoethyl ether)-N,N,N,′N′-tetraacetic acid and incubated at 37°C for 15 minutes. During incubation, islets were gently aspirated into and released from a plastic pipette. This procedure yielded suspensions largely composed of single cells with a few aggregates of three to five cells. Fluorescein diacetate uptake6 studies revealed that a mean (±SE) of 70±5 percent of the cells were viable.
Measurement of Uptake
The dispersed cells from 3500 islets were incubated in 0.7 ml of IgG solution containing a final concentration of 2 mM [14C]urea (0.5 μCi per micromole) as an intracellular-space marker at 37°C for 20 minutes, followed by incubation at 15°C for 10 minutes. The uptake of glucose analogues was determined according to a modification of the method of Gorus et al.7 For 3-O-methyl-β-D-glucose, 50 μl of 1 M D-glucose containing 10 mM EDTA and 0.1 percent sodium dodecyl sulfate (pH 8.0) was placed in the bottom of 400-μl microfuge tubes. This solution was overlaid with 150 μl of a dibutyl phthalate–dinonyl phthalate (4:1) mixture. After a 30-second centrifugation in a Beckman microfuge, 50 μl of phosphate-buffered saline containing 2 mM [14C]urea (0.5 μCi per micromole) and 20 mM 3-O-β-D-[3H]methyl-glucopyranoside (5 μCi per micromole) was layered over the dibutyl phthalate–dinonyl phthalate phase. The tubes were then incubated for 20 minutes at 15°C. Uptake was initiated by adding 50 μl of the cell suspension incubated with urea to the 50-μl phosphate-buffered saline phase containing labeled glucose analogue and urea. Uptake was terminated by centrifuging the cells for 15 seconds so that they passed through the dibutyl phthalate–dinonyl phthalate layer into the 1 M glucose10 mM EDTA0.1 percent sodium dodecyl sulfate solution. Then, 50 μl of the supernatant was removed from each tube for calculations of specific activity, and 35 μl of the 1 M D-glucose containing 10 mM EDTA and 0.1 percent sodium dodecyl sulfate was removed for the determination of uptake in a Beckman LS 5801 liquid scintillation counter. The uptake was measured in duplicate at the times indicated in each experiment. The results were calculated as disintegrations of each isotope per minute with the resident calculation program of the counter and were expressed in millimoles per liter of islet space. Corrections for extracellular space as judged by the measurement of L-glucose at time zero were 19, 22, and 28 percent of the total urea space in experiments performed in buffer alone, buffer containing IgG from normal subjects, and buffer containing IgG from patients with IDDM, respectively. Approximately 103 islet cells per milligram of IgG were present during these incubations. The initial rates of 3-O-methyl-β-D-glucose uptake were measured at 3, 6, and 15 seconds. The uptake of L-[l-3H]glucose and L-[3,4,5-3H]leucine was measured by the same procedure. The results are expressed as the means ±SEM of the values at each time point for all IgG fractions of each type tested.
Adsorption of IgG Preparations
Adsorption studies were carried out to determine whether the inhibition of glucose transport mediated by the IgG fractions was abolished by preincubation with various tissues containing glucose transporters similar or dissimilar to those of beta cells. We prepared unsealed red-cell ghosts,8 liver plasma membranes,9 and kidney brush-border specimens.10 The IgG preparations were diluted to a concentration of 4 mg per milliliter of phosphate-buffered saline and incubated with dispersed islet cells from 40 rats (approximately 20,000 islets), 20 mg of liver plasma membrane protein, 20 mg of rat red-cell ghost protein, 20 mg of kidney brush-border membrane, or buffer alone for three hours at 4°C. The mixtures were then centrifuged once at 800×g for 5 minutes and twice at 50,000×g for 10 minutes to remove islet cells or membranes. The supernatants were carefully removed and stored at 4°C overnight, after which they were added to dispersed rat islet cells. After incubation the cells were assayed for 3-O-methyl-β-D-glucose uptake as described above.
Statistical Analysis
The results were decoded, segregated according to diagnosis, and compared by an unpaired two-tailed t-test.
Results
Characteristics of the Study Groups
The sex, age, diagnosis, treatment, and for the patients with IDDM, islet-cell antibody status of the study group are shown in Table 1Table 1
Characteristics of the Diabetic Patients and Normal Subjects.*.Glucose Uptake by Islet Cells
As shown in Figure 1Figure 1
Time Course of the Uptake of 3-O-Methyl-β-D-glucose (Open Circles) and L-Glucose (Solid Circles) by Dispersed Rat Islet Cells., the uptake of 3-O-methylβD-glucose by islet cells was consistent with facilitated diffusion. The time required for half-maximal uptake was less than 15 seconds, and equilibration with the extracellular 3-O-methyl-β-D-glucose concentration was more than 90 percent complete in one minute. Since all known glucose transporters are stereo-specific, L-glucose should be excluded from intact cells.7 , 11 , 12 The uptake of L-glucose at time zero was 19.2 percent of the urea space. It increased slightly for the first six seconds and did not change thereafter, thus providing evidence that the permeability barrier of the islet cells was intact (Fig. 1). The uptake of L-glucose was determined at time zero in subsequent studies to provide an index of the extracellular space and cellular integrity.Effects of IgG from Diabetic Patients and Normal Subjects on 3-O-Methyl-β-D-glucose Uptake by Islet Cells
An initial comparison of the effects of the IgG fractions from the first 8 patients with IDDM and 11 normal subjects studied without knowledge of the diagnosis revealed significant inhibition of 3-O-methyl-β-D-glucose accumulation at 3, 6, 15, and 30 seconds (P<0.01), but not at one minute or later. As would be expected in a facilitated-diffusion transport system that was not completely inhibited, the inhibition was greatest at the earliest points. Subsequent comparisons in additional subjects were therefore restricted to the 3-, 6-, and 15-second time points, because they provided the most accurate estimation of the initial rate of uptake. The IgG fractions from 27 patients with new-onset IDDM significantly inhibited (P<0.001 at each time point) the transport of 3-O-methyl-β-D-glucose in the dispersed rat islet cells as compared with the IgG fractions from 28 normal subjects (Fig. 2Figure 2
Effects of IgG Fractions from Normal Subjects (Open Circles) and Patients with IDDM (Solid Circles) on the Uptake of 3-O-Methyl-β-D-glucose and L-Leucine by Dispersed Rat Islet Cells.). Progressive dilution of IgG resulted in progressive loss of this inhibitory activity.To determine whether the inhibition was restricted to 3-O-methyl-β-D-glucose transport or whether the inhibition of 3-O-methyl-β-D-glucose transport was due to a nonspecific alteration in the permeability of the islet cells exposed to islet-cell antibodies, the uptake of L-leucine (Fig. 2) was measured after the incubation of islet cells with the coded IgG fractions from 9 patients with IDDM and 15 normal subjects. The uptake of L-leucine into islet cells incubated with IgG fractions from patients with IDDM and normal subjects was virtually identical.
Figure 3Figure 3
Initial Rates of 3-O-Methyl-β-D-glucose Uptake by Rat Islet Cells in the Presence of IgG from Each Member of the Three Study Groups. shows the initial rates of 3-O-methyl-β-D-glucose uptake in the presence of the IgG fractions from the 28 normal subjects, 27 patients with IDDM, and 5 patients with NIDDM. In islet cells incubated with IgG from patients with IDDM, the rates of 3-O-methyl-β-D-glucose transport at 3, 6, and 15 seconds were approximately 50 percent lower than the rates in islet cells incubated with IgG from normal subjects (P<0.001). The rates in islet cells incubated with IgG from patients with NIDDM and other autoimmune diseases (two patients with Graves' disease and one with systemic lupus erythematosus; data not shown) were similar to the rates of uptake in the presence of IgG from normal subjects. The reproducibility of the IgG effect on the initial rates of 3-O-methyl-β-D-glucose uptake was evaluated two to five times with IgG fractions from four normal subjects and four patients with IDDM. The standard deviation of the mean of replicate determinations was ±1.8 mmol per minute per liter of islet space for IgG from the four patients with IDDM and ±0.8 mmol per minute per liter of islet space for IgG from the four normal subjects.Examination of the dependence of the glucose concentration on 3-O-methyl-β-D-glucose uptake by the islet cells used in this study revealed two kinetically distinct facilitated-diffusion transporters: one with an apparent Michaelis constant (Km) of 18 mM and another with an apparent Km of 1.5 mM (Johnson JH: unpublished data). The transporter with a Km of 18 mM is kinetically similar to the liver transporter, and studies with an antibody to the liver transporter indicate that in the pancreas, the antibody is localized to beta cells.9 , 13 To test whether the IgG fractions from patients with IDDM affect glucose transport by preferentially inhibiting the beta-cell transporter with a Km of 18 mM or the transporter with a Km of 1.5 mM, the dependence of the glucose concentration on the uptake of 3-O-methyl-β-D-glucose was examined in islet cells incubated with IgG from three patients with IDDM and three normal subjects. Figure 4Figure 4
Effects of IgG from Normal Subjects (Open Circles) and Patients with IDDM (Solid Circles) on Glucose Uptake by Rat Islet Cells with Various Concentrations of Glucose. shows that the IgG fractions from the patients with IDDM inhibited the beta-cell transporter with a Km of 18 mM without affecting the transporter with a Km of 1.5 mM. The presence of IgG fractions from the normal subjects did not alter these kinetics. Furthermore, the IgG from the patients with IDDM decreased the maximal velocity of uptake without altering the Km. These data further support an interaction between the IgG and beta-cell glucose transporters.Effects of Insulin and Insulin Antibodies on 3-O-Methyl-βD-glucose Uptake by Islet Cells from Normal Rats
The IgG fractions from the patients with IDDM, all of whom had been treated with insulin for several weeks, may well have contained insulin antibodies14 or autoantibodies15 not present in the IgG fractions from the normal subjects. To exclude the possibility that such antibodies might influence the uptake of glucose, 3-O-methyl-β-D-glucose uptake was measured in the presence and absence of guinea pig anti-insulin serum. The guinea pig anti-insulin serum had no effect on the uptake of 3-O-methyl-β-D-glucose by islet cells; the uptake rate was 9.8±0.8 mmol per minute per liter of islet-cell space in the presence of anti-insulin antibody (10 μg per milliliter; n = 3), as compared with 11.3±0.8 mmol per minute per liter of islet space in the presence of buffer. We also tested the possibility that insulin copurified with insulin-binding antibodies might bind to islet cells and alter glucose transport. The uptake rate of cells pretreated for 20 minutes with 1 mU of porcine insulin was 10.0±1.2 mmol per minute per liter of islet space (n = 3), as compared with 11.3±0.8 mmol per minute per liter of islet space in cells preincubated with buffer. Islet cells pretreated simultaneously for 20 minutes with the same amounts of insulin and anti-insulin antibody took up 3-O-methyl-β-D-glucose at a rate of 10.2±1.1 mmol per minute per liter of islet space. Therefore, insulin, anti-insulin antibody, or a combination of the two had no effect on the uptake of 3-O-methyl-β-D-glucose by islet cells.
Effects of IgG on 3-O-Methyl-β-D-glucose Uptake by Erythrocytes and Hepatocytes
Recent evidence indicates that several structurally distinct facilitated-diffusion glucose transporters exist in mammalian tissues. The glucose transporter from erythrocytes, adipocytes, and brain cells differs from the transporter found in liver cells,16 whereas antibodies to a synthetic 13–amino acid peptide from the predicted sequence of the C-terminal region of the 55-kd glucose transporter from rat liver reacts with a transporter expressed in beta cells.13 , 16 The IgG fractions from four patients with IDDM did not inhibit 3-O-methyl-β-D-glucose uptake into human erythrocytes, but it also failed to inhibit uptake by rat hepatocytes, despite the apparent similarity of liver and beta-cell glucose transporters (Fig. 5Figure 5
Effects of IgG Fractions from Normal Subjects (Open Circles) and Patients with IDDM (Solid Circles) on the Uptake of 3-O-Methyl-β-D-glucose by Human Erythrocytes and Rat Hepatocytes.).Effects of Preincubation of IgG from Patients with IDDM with Various Tissues on the Inhibition of Glucose Transport by IgG
Adsorption experiments were performed to determine whether the inhibitory effect of the IgG fractions from the patients with IDDM on 3-O-methyl-β-D-glucose transport in islet cells could be abolished by preincubation with cells or cell membranes that either do or do not express the glucose transporter shared by liver and beta cells. A total of 4 mg of IgG from four patients with IDDM was incubated with cells from 20,000 islets, 20 mg of hepatocyte membranes (cells that normally share the same glucose transporter), and 20 mg of plasma membranes from rat erythrocytes and renal-tubular-cell brush borders (neither of which contain this transporter). As shown in Figure 6Figure 6
Effect of Preincubation of IgG from Four Patients with IDDM with Cells or Cell Membranes from Islets, Liver, Erythrocytes, or Kidney Brush Border on 3-O-Methyl-β-D-glucose Uptake by Islet Cells., the inhibitory effect was abolished by incubation of the IgG with islet cells and hepatocyte membranes but not by incubation with erythrocytes or brush-border membranes.Discussion
The demonstration by Kanatsuna et al.1 that immunoglobulins from patients with new-onset IDDM inhibit glucose-induced insulin secretion from perfused rat islets raised the possibility that a specific function of islet cells might be inhibited by such immunoglobulins. Because of our subsequent demonstration that pancreatic islets isolated from BB/W (BioBreeding/Worcester) rats on the first day of their autoimmune diabetes had no insulin secretory response to glucose but a normal response to arginine,3 we considered that anti-beta-cell antibodies might be directed against either the glucose transporter or another protein involved in glucose transport. The results of this study are consistent with this possibility, in that the IgG fraction of serum from patients with new-onset IDDM inhibited the initial rates of 3-Omethyl-β-D-glucose uptake by dispersed rat islet cells but had no effect on the uptake of L-leucine. It is unlikely that these results were due to cytotoxic effects of the IgG fractions, because nonviable cells do not pass through the dibutyl phthalate layer of the assay tubes during centrifugation and are therefore not measured. Furthermore, the fact that L-glucose permeability was not affected by the IgG fractions from the patients with IDDM indicates that the cellular integrity was maintained.
The IgG fractions from the patients with IDDM did not affect 3-O-methyl-β-D-glucose uptake by hepatocytes or erythrocytes. Thorens et al.16 have shown that the liver glucose transporter differs substantially from the glucose transporter of erythrocytes, whereas it is similar if not identical to that of the beta cells. Antibodies to a synthetic peptide deduced from the sequence of the liver transporter recognize a protein on islet beta cells, suggesting that these transporters may have similar structures.13 Moreover, a complementary-DNA library prepared from pancreatic islets from normal rats contained clones with a sequence identical to that of the liver glucose transporter in rats (Johnson JH, Newgard CB, Milburn J, Lodish H, Thorens B: unpublished data). Our results indicate that islet cells have a transporter with kinetic properties similar to those of the liver transporter and that the IgG fractions from the patients with IDDM exerted their inhibitory effects preferentially on the transporter with a Km of 18 mM. The fact that the inhibitory effect on 3-O-methyl-β-D-glucose uptake by islet cells was abolished by preincubation with both islet cells and hepatocyte membranes, but not by preincubation with preparations of erythrocytes or kidney brush-border membranes, provides support for the interpretation that the liver and beta-cell transporters are similar and is consistent with but does not prove the antigen–antibody hypothesis of glucose-transport inhibition. The failure of IgG from patients with IDDM to inhibit glucose transport by hepatocytes may indicate an excess of glucose transporters relative to the concentration of putative antibody.
Antibodies to at least three islet antigens have been found in the serum of patients at the time of the diagnosis of IDDM. These antigens are the 64-kd membrane protein reported by Baekkeskov et al.,17 cytoplasmic islet-cell antigens,18 and insulin.15 There is no reason to believe that the 64-kd protein is related to the 55-kd glucose transporter. Tests for cytoplasmic autoantibodies were negative in almost half the patients with IDDM whose IgG inhibited 3-O-methyl-β-D-glucose transport activity. Although insulin autoantibodies were not assayed in these patients, we established that incubation of islet cells with insulin antibodies had no effect on the uptake of 3-O-methyl-β-D-glucose.
The functional implications of a 50 percent reduction in 3-O-methyl-β-D-glucose uptake by islet cells are unclear. Glucose-transport capacity far exceeds the capacity to phosphorylate glucose,19 suggesting that this degree of interference with glucose transport is probably not sufficient to affect insulin secretion in vivo. However, the IgG concentrations used in this study were 20 percent of those, on a basis of the protein-to-cell ratio, used by Kanatsuna et al.1 to demonstrate IgG-induced inhibition of insulin secretion by islets. Also, 3-O-methyl-β-D-glucose uptake was measured in a mixture of islet cells that included non—beta cells, in which 3-O-methyl-β-D-glucose uptake differs kinetically from that of beta cells7 and the liver glucose transporter is not expressed. This may well have minimized the inhibition. Nevertheless, we consider it unlikely that the antibody-induced inhibition of glucose uptake in vitro caused the loss of glucose-stimulated insulin secretion in vivo.
Even though there is no reason to assume that the putative glucose-transporter antibodies are cytotoxic to beta cells, they may initiate a chain of events leading to the cessation of glucose-stimulated insulin release. Alternatively, they may be an epiphenomenon, an immune response to the release of beta-cell antigens after the destruction of beta cells by an unidentified process.
In summary, we found that purified IgG from patients with newly diagnosed IDDM inhibited the initial uptake of 3-O-methyl-β-D-glucose in rat islet cells. This effect was abolished by preincubation of the IgG with islets and with hepatocyte membranes, both of which contain the same glucose transporter. These results are consistent with but do not prove an antigen–antibody reaction with either the beta-cell glucose transporter or a functionally related protein.
Supported by grants from the Juvenile Diabetes Foundation (187417), the Department of Veterans Affairs, the National Institutes of Health (AM-02700), and the Greenwall Foundation.
We are indebted to Drs. J.D. McGarry and M. Daniels for the preparation of hepatocytes, to Dr. M. Appel and Mr. J. O'Neil of the University of Massachusetts Medical School for suggesting modifications to our islet-preparation method, and to Dr. Dennis Stone for his interest and advice.
Source Information
From the Center for Diabetes Research, Gifford Laboratories, University of Texas Southwestern Medical Center (J.H.J., M.A., R.H.U.), the Department of Medicine, Veterans Affairs Medical Center (J.H.J., K.McC., R.H.U.), and the Department of Physiology, University of Texas Southwestern Medical School (B.P.C.), Dallas. Address reprint requests to Dr. Johnson at the Center for Diabetes Research — 8854, 5323 Harry Hines Blvd., Dallas, TX 75235.
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Marcia F. McInerney, Jeffrey Burkey, Liping Guan, Jeffrey C. Flynn, Katherine I. Oravecz, Charles A. Janeway Jr. (2000) An islet-specific CD8+ T cell hybridoma generated from non-obese diabetic mice recognizes insulin as an autoantigen. Diabetes Research and Clinical Practice 47:3, 151-168
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Elena Sarugeri, Nicoletta Dozio, Franco Meschi, Matteo Rocco Pastore, Ezio Bonifacio. (1999) Cellular and Humoral Immunity against Cow's Milk Proteins in Type 1 Diabetes. Journal of Autoimmunity 13:3, 365-373
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