spontaneous Hypoglycemia Associated With ... - Diabetes Care

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pontaneous Hypoglycemia Associated With Autoimmunity Specific to Human Insulin

IVO SKLENAR, MD, TERENCE J. WILKIN, MD, JOSE-LUIS DIAZ, MD, PETER ERB, PhD, AND ULRICH KELLER, MD

A 55-yr-old woman with a history of Graves' disease experienced attacks of postprandial hypoglycemia for 6 yr. An insulinoma could not be confirmed by repeated fasting tests and by surgical pancreas revision. Extracted pancreatic insulin was chemically normal. Fasting plasma total insulin (1.22 nM = 183 jjiU/ml) and proinsulin (0.48 nM) were elevated and greatly increased after oral glucose. Glucoseclamp studies revealed delayed insulin clearance. Plasma free-insulin levels were normal. Insulin-binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay with human insulin as ligand but not with pork or beef insulins. Analysis with a modified ELISA suggested a monotypic and monoclonal human insulin autoantibody, which showed a restriction to the \-light chain. T-lymphocytes (predominantly helper) demonstrated increased responsiveness to beef, pork, and human insulins by proliferation assay. A T-lymphocyte line showed exclusively human insulin specificity. All this indicated cellular and humoral anti-human insulin autoimmunities. Clinically, the cause of hypoglycemia associated with elevated total insulin and proinsulin was misdiagnosed as atypical insulinoma. The study of total and free plasma insulin levels and sensitive antibody assays specific to human insulin were necessary to correctly diagnose autoimmune hypoglycemia. Diabetes Care 10:152-59, 1987

D

espite its low molecular weight of ~6000, insulin is a potent immunogen. Exogenous administration of insulin in some individuals can lead to the formation of insulin antibodies capable of binding and releasing large amounts of the hormone. Hypoglycemic attacks may result (1). The first report of spontaneous hypoglycemia without evidence of exogenous insulin administration was published by Hirata et al. (2) in 1970. Since then, several similar cases have appeared in the literature (3-7). Lack of evidence of previous insulin administration and exclusion of other causes of hypoglycemia together with high levels of total serum immunoreactive insulin led to the conclusion that spontaneous autoimmunity was the cause. However, the erroneous or clandestine administration of insulin may also lead to hypoglycemia and insulin-antibody formation (8,9), and where there is doubt, the triad of low plasma glucose, high immunoreactive insulin, and suppressed C-peptide can help to identify exogenous insulin administration as a cause (8,9). However, in the presence of insulin antibodies, the C-peptide may be elevated even in factitious hypoglycemia (9). In comparison, the available data in the literature indicate that the

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C-peptide levels in autoimmune hypoglycemia are either normal or lowered (7). We describe a 55-yr-old woman with recurrent bouts of severe spontaneous hypoglycemia who had never received insulin, whose personality did not show a disposition to insulin self-administration, who had C-peptide levels in the normal fasting range during hypoglycemia, and who developed human insulin—specific IgG antibodies before human insulin was available in Switzerland. Studies of her humoral and cellular immune responses suggested that both B- and T-lymphocytes were sensitized to insulin. A sensitive enzyme-linked immunosorbent assay (ELISA) designed to detect autoantibodies specific for human insulin was necessary to confirm the diagnosis clinically. MATERIALS AND METHODS

Antibody studies. Insulin antibodies were measured at various points during this investigation by three methods. 1. Polyethylene glycol (PEG) precipitation method (10). Varying doses (0.1-26 ng) of either 125I-labeled pork or beef insulin (specific radioactivity 23-30 |xCi/u,g; Novo, Bags-

DIABETES CARE, VOL. 10 NO. 2, MARCH-APRIL 1987

HYPOGLYCEMIA AND AUTOIMMUNITY SPECIFIC TO HUMAN INSULIN/I. SKLENAR AND ASSOCIATES

vaerd, Denmark) were added in duplicate to 40 (xl of serum, incubated for 2 h at 37°C, and then incubated overnight at 4°C. PEG 6000 (Merck, Darmstadt, FRG) at 20% final concentration was used to precipitate the antibody-bound tracer, which was washed and counted in a 7-counter (Packard, Downers Grove, IL). Maximum binding (percent total cpm) was achieved with 0.2-ng tracer in most cases, as in other studies (11), and this quantity was subsequently used throughout the study. Background binding was subtracted from each sample with a fetal calf serum control. A similar PEG precipitation assay was developed with 125I-human insulin (courtesy of Dr. Mary Root, Lilly, Indianapolis, IN). 2. Rocket immunoelectrophoresis (12). The IgG- and IgMspecific binding capacities of labeled bovine insulin were measured by radioimmunoelectrophoresis at the Novo Research Institute. 3. ELISA. A sensitive and IgG-specific ELISA was used to study binding to highly purified human, pork, and beef insulins (courtesy of Dr. Mary Root) (13). The results were expressed as percentage of the binding of a pooled reference serum to the appropriate insulin. The interassay variation was 10%, and the sensitivity was 20 times greater than that of the PEG precipitation radioimmunoassay (RIA) for the measurement of insulin autoantibodies. Antibody-adsorption studies. The serum was adsorbed in a dose-dependent fashion with a fixed mass of Sepharose beads to which increasing amounts of insulin were attached. In brief, either highly purified human or pork insulin was covalently coupled to cyanogen bromide-treated Sepharose 4B beads (Pharmacia, Uppsala, Sweden) to provide a concentration range from 0.04 to 40,000 |xg/ml neat serum. The beads, and the 1.5-ml Microfuge tubes (Eppendorf, Hamburg, FRG) into which they were pipetted, were blocked with 1.0% bovine serum albumin. Beads (28.6 mg dry wt) were incubated overnight in constant motion at 4°C with 700 |xl serum at a dilution of 1:30 in phosphate-buffered saline Tween (Merck, 0.05%) at pH 7.4. The incubate was then centrifuged and the supernatant tested for antibodies against human and pork insulins in the ELISA. Total insulin, C-peptide, and pro insulin were determined by RIA at the Novo Research Institute (14-16). Free-insulin levels were measured after PEG extraction of antibodies from the plasma (17). Cellular immunology studies. Peripheral blood lymphocytes (PBL) were obtained by Ficoll-Paque centrifugation (Pharmacia). Lymphocyte transformation experiments with 100 |xg/ml of human, pork, or beef monocomponent zinc insulin (Novo Monotard HM, Monotard MC, Ultralente MC); 10 |xg/ml of tetanus toxoid; or 100 |xg/ml purified protein derivative (PPD) (Institut Serotherapique et Vaccinal, Bern, Switzerland) were performed. Cells (1 X 105) were cultured for 7 days in a flat-bottomed Micro Test II plate (Falcon, Oxnard, CA) with Dulbecco's modified Eagle's medium with antibiotics, amino acids (18), and 10% inactivated pooled human AB serum. During the last 8 h of culture, the cells were pulsed with 0.25 |xCi/well of 125I-UdR (Amersham, Amersham, Buckinghamshire, UK), harvested with a Titer-

tek Cell Harvester (Flow, Irvine, Ayrshire, UK), and the counts per minute were measured in the -y-counter. Stimulation indices (cpm antigen/cpm no antigen) were calculated from quadruplicate cultures. Lymphocyte long-term cultures (LTC) were induced with various zinc insulin species as described for other antigens (18). Briefly, 1 X lOVwell PBL were cultured with 10-100 jxg/ml of human, pork, or beef zinc insulin in Micro Test plates. On the 10th day, half of the medium in each well was replaced with fresh medium containing 10% of a crude human interleukin 2 (IL-2) prepared as described elsewhere (19). On the 14th day, cultures showing the highest proliferation on microscopic examination were further expanded in more wells with 3 X lOVwell irradiated (3000 rads) autologous adherent cells (AC), IL-2, and antigen. Tissue culture flasks (Falcon) were used to obtain more cells for further analysis. Antigen-specific proliferation was tested with 1 X 10s cells in duplicate, which were cultured with 3 X 104 autologous AC and various insulins or PPD. After 72 h the incorporation of 125I-UdR was measured. In an indirect immunofluorescence test the lines were labeled with monoclonal antibodies directed at the helperinducer T-lymphocyte surface marker T4 (Leu 3a, Becton Dickinson, Mountain View, CA) and cytotoxic-suppressor marker T8 (Leu 2b) and analyzed with goat anti-mouse IgFITC conjugate and the immunofluorescence microscope. Metabolic clearance of insulin and insulin action on glucose metabolism was determined with the glucose-clamp technique (20). Insulin clearance was calculated by dividing the insulin infusion rate during the glucose clamp by the increase in total plasma insulin concentration (21). CASE REPORT

S.M., a 55-yr-old Swiss woman, was referred for evaluation of spontaneous hypoglycemia. She was nondiabetic with no known or apparent psychiatric disorder. Her occupation was not related to health care, and neither alcohol nor drug abuse, which could cause enhancement of insulin secretion (22), was present. In 1971, Graves' disease was diagnosed, and she was treated with carbimazole for 1 yr. She subsequently received 131I therapy. In 1978, impaired glucose tolerance with symptomatic late hypoglycemia (2.8 mM 180 min after 50-g oral glucose) was noted. During the next few years, intermittent spells of weakness, sweating, and tachycardia occurred. They were frequently precipitated by exercise, improved after food intake, and usually occurred 1—3 h after meals. In 1981, the patient was admitted to another hospital as an emergency with documented hypoglycemia (blood glucose 1.8 mM) occurring in the late afternoon, somnolence, and hemiparesis, which improved after intravenous glucose. The next day, she experienced severe hypoglycemia while under close supervision; her blood glucose was 2.8 mM, total plasma insulin concentration drawn simultaneously was 0.43 nM (65 fxU/ml; normal, 0.033-0.133 nM = 5-20 |xU/ml), and C-peptide was 0.32 nM (0.97 ng/ ml; normal, 0.29-0.7 nM = 0.88-2.12 ng/ml).


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plasma C-peptide 0.44 nM (1.33 ng/ml), free C-pepttde 0.466 nM (1.4 ng/ml), and blood glucose 4.3 raM. The patient remained well for 5 yr after surgery on a regimen of fractionated meals. Hypoglycemic attacks were frequently documented by the patient via Chemstrips. Attacks were reduced in severity and number to one to three per month.

FAST

15 ,

1.0 Insulin (nmol/l)

RESULTS

Antibody studies. Total plasma insulin after an overnight fast before surgery was 1.22 nM (183 (xU/ml), whereas free insulin was 0.043 nM (6.5 |xU/ml). Plasma proinsulin was 0.48 nM (normal, 0-0.036 pM). Plasma C-peptide was similar before and after PEG extraction (0.39 nM = 1.18 ng/ ml and 0.4 nM = 1.2 ng/ml, respectively). Total plasma insulin, proinsulin, C-peptide, and glucose concentrations during an oral glucose tolerance test are shown in Fig. 2. Basal levels of blood glucose and C-peptide were

0.5 •

Glucose (mmol/l)

ORAL GLUCOSE TOLERANCE TEST

2-

02^ /30HB (mmol/l)

) _| Insulin (nmol/l)

0J ? 0

24

48

72 Hours

FIG. 1. Seventy-two-hour fasting experiment showed initial total plasma insulin concentration of 1.25 nM (188 |xU/ml), which decreased to 0.6 nM (90 |AU/ml) after 12 h of fasting. No fasting hypoglycemia was noted, and fasting hyperketonemia developed. Starting plasma total insulin level was similar to that in oral glucose tolerance test (OGTT), but it decreased subsequently during fast, whereas it increased dramatically during OGTT (see Fig. 2). Serum binding capacities of pork and beef insulins were normal, and IgG and IgM antibodies binding pork and beef insulins were not detected. An insulinoma was suspected because of negative antibody results, although atypical in that there was never a fasting hypoglycemia documented. Repeated fasting tests up to 72 h did not provoke hypoglycemia but provoked fasting hyperketonemia (Fig. 1). Because of the severity of hypoglycemic attacks, exploratory surgery and partial pancreatectomy (tail resection) were performed in 1981. The suspected insulinoma was not found, and the remainder of the pancreas was free of tumor by careful palpation. High total insulin but normal free-insulin levels measured with PEG extraction (17) persisted postoperatively (14). In 1983, overnight fasting total plasma insulin was 1.306 nM (196 ixU/ml), free insulin 0.031 nM (4.6 |xU/ml), and blood glucose 5.3 raM. In 1984, the fasting total insulin was 0.42 nM (63 M-U/ml), free insulin 0.033 nM (5 jiU/ml), total

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CONTROLS (Xtsd)

C-Peptide (nmol/l)

Proinsulin (nmol/l)

Glucose (mmol/l)

30

60

90

120

240 Minutes

FIG. 2. Oral glucose tolerance test (75 g) demonstrated marked hyperinsulinemia (total insulin; 14) and hyperproinsulinemia (16). C-peptide levels were normal in basal state and increased after oral glucose (15), suggesting insulin hypersecretion after glucose challenge (insulin, proinsulin, and C-peptide levels were determined by Novo, Bagsvaerd, Denmark).


HYPOGLYCEMIA AND AUT0IMMUN1TY SPECIFIC TO HUMAN INSULIN/I. SKLENAR AND ASSOCIATES

TABLE 1 Binding of '"I-labeled pork and beef insulins by patient's serum and proliferation of peripheral blood lymphocytes after stimulation with human, pork, and beef zinc insulins Healthy controls Age (yr) (N) Percent bound pork (range) Percent bound beef (range)

38 ± 17 (14) 0.7 ± 0.2 (0-2.7) 0.9 ± 0.4 (0-5.1)

Stimulation index§ Human insulin Pork insulin Beef insulin

9.6 ± 1.6 9.8 ± 1.8 14.6 ± 2.3

Tetanus toxoid (N)

10.8 ± 2.9 (13)

Insulin-treated diabetics

42 ± 15 (23) 10.0 ± 2.1 (0.4-34.4)1 20.4 ± 4.4 (0.5-65.6)1: 8.4 10.2 16.4 19.9

± ± ± ±

1.9 1.9 2.8 5.5 (23)

Patient S.M.

55 (3)* 1.5 ±0.6(0-2.7) 0.1 ± 0.06(0-0.2) 70.2 ± 23.7 59.1 ± 21.4 136.9 ± 24.5 4.8 ±0.7 (4 cultures)

Values are means ± SE; age, mean ± SD. "Serum was tested between 1981 and 1983. tP < .001. $P < .01, compared with controls, by a two-sample Student's t test. §Stimulation index = cpm antigen/cpm Nil (Nil, cells cultured in medium with no antigen).

normal, whereas insulin and pro insulin levels were elevated. After oral glucose, total insulin and proinsulin levels increased markedly above those of controls. Hypoglycemia developed 240 min after oral glucose challenge, and the Cpeptide level was in the range of normal controls. This might be why the C-peptide level was also found normal during the episode of spontaneous postprandial hypoglycemia. Insulin clearance determined with the glucose-clamp technique was 4-4 times lower than that in normal subjects. The insulin infusion rate during the clamp was 0.15 nmol • m" 2 • min" 1 in controls and in the patient. The average plasma insulin increase in controls was 0.25 nM, which corresponds to an insulin clearance of 0.6 L • m" 2 • min" 1 . In contrast, S.M. demonstrated an average increase of total plasma insulin of 1.1 nM, yielding a calculated insulin clearance of only 0.136 L • m" 2 • min" 1 . Glucose requirements during the clamp were similar to those of normal controls, indicating unimpaired insulin sensitivity of glucose metabolism. The histology of the pancreas after surgery revealed no evidence for insulitis. Amino acid content of extracted pancreatic insulin, determined with HPLC, demonstrated normal composition. Table 1 shows the proportion of 125I-labeled beef and pork insulin precipitable by PEG in the sera from 14 controls and 23 diabetics treated with crystalline beef, pork, and soluble pork monocomponent insulins (Lente and Actrapid, Novo) compared with binding in serum from S.M. Significantly higher binding of pork (P < .001 by 2-sample Student's t test) and of beef (P < .01) insulins was found in the sera of diabetics compared with 14 healthy controls. Patient S.M. was tested on three occasions between 1981 and 1983, and the pork and beef insulin binding of her serum was always found to be within the normal range. Similarly, no IgM and IgG insulin antibodies were detectable with radioimmunoelectrophoresis with beef insulin as a tracer (Novo). In addition, the sera of S.M. and controls were incubated

with native human and pork insulins in the ELISA and with labeled human and pork insulins in the PEG precipitation RIA. S.M.'s binding was specific for human insulin in both assays. With RIA, her serum bound 49.3% (percent of total added cpm) human but only 3.7% of the added pork insulin tracer. In comparison, S.M.'s serum bound 352% (percent of reference serum binding) human insulin and only 2.6% pork insulin in IgG-specific ELISA. The results of adsorption studies of S.M.'s serum with human and porcine insulins are shown in Fig. 3. Human insulin was able to adsorb 100% of the binding activity to human insulin at a concentration of 103 |xg insulin/ml serum, whereas pork was able to remove barely 5% of binding to human insulin at the highest concentration of 105 |xg insulin/ ml serum. The Scatchard analyses of serum from S.M. and from an insulin-treated diabetic subject were performed (data not shown). The plot for S.M. was linear, yielding a human insulin-binding capacity of 2.1 X 10~7 M (1.2 X 103 ng/ ml = 30,000 M-U/ml) and an affinity constant of 5.4 X 106 L/M (9.3 X 10~4 ml/ng), whereas the plot for the diabetic serum was continuously curvilinear. The human insulin tracer binding in S.M.'s serum was interpreted as a single binding reaction. Analysis with a modified ELISA revealed a monotypic human insulin autoantibody (\)> which suggested monoclonality. Antibody affinity and binding capacity at all points during a prolonged 75-g oral glucose tolerance test revealed no significant variations (data not shown). Cellular immunology studies. In contrast to the failure of S.M.'s serum to bind pork and beef insulins, the transformation response to all three insulin species was significantly greater in lymphocytes from S.M. than in those from controls and diabetics. The highest proliferation of S.M.'s lymphocytes was obtained with human and beef insulins, but differences between the three insulin species were not significant (Table 1).


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10°

101

FIG. 3. S.M.'s serum preadsorbed with in* creasing concentrations of highly purified human (A) and pork (A) insulins attached to fixed mass of Sepharose beads, assayed against human (solid line) and pork (dashed line) insulins in the enzyme-linked immunosorbent assay.

10"

/Ug Insulin/ml serum

To identify restriction among proliferating lymphocytes, LTCs were induced with human and beef insulins. LTCs showing antigen-specific proliferation after restimulation with autologous ACs and insulin were analyzed with indirect immunofluorescence for the presence of T-lymphocyte markers T4 (helper-inducer) and T8 (suppressor-cytotoxic) characterized by monoclonal antibodies (Leu 3a and Leu 2b). One T-lymphocyte line, which was obtained by induction with 10 (xg/ml of human insulin, responded significantly only to restimulation with a low dose of human insulin (1 |xg/ml) but not with pork or beef insulin (Table 2). The cells failed to respond to stimulation with a high dose of human insulin (10 and 100 |xg/ml) and consisted predominantly of T-lymphocytes carrying the T4 marker of helper-inducer cells (85%)

and few T 8 + (cytotoxic-suppressor) cells (10%). A second line from S.M., induced by human insulin, reacted only with a low dose of human insulin but proliferated with both low and high doses of pork and beef insulins (not shown). It contained 65% of T 4 + and 14% T 8 + cells. A third line from S.M., induced with a high dose of beef insulin (100 (xg/ml), responded unusually vigorously to restimulation with 100 |xg/ml of human, pork, and beef insulins (Table 2). In contrast, a line induced with 100 (xg/ ml pork insulin from L. W., a 42-yr-old type I diabetic subject treated with pork insulin and with similarly low binding of his serum of pork and beef insulins as S.M., responded only to stimulation with beef insulin (Table 2). In summary, there was one exclusively human insulin-

TABLE 2 Proliferation of insulin-induced T-lymphocyte lines of patient S.M. and type I diabetic patient L.W. and percent of T-lymphocyte surface markers Phenotype of cells (%)

Mean cpm after stimulation Patient

Inducer

Human insulin

Pork insulin

Beef insulin

PPD/Nil§

T4 +

T8 +

S.M.

10 (ig/ml human 100 M-g/ml beef 100 M-g/ml pork

1238 (6)* 8361 (8) 1232 (2)

683 (3) 10,257 (10) 1210 (2)

585 (3) 11,614 ( l l ) t 3014 (5)$

158 (0)/204 446 (0)/1069 NT/625

85 NT 72

10 NT 23

LW.

NT, not tested. Stimulation index = cpm Ag/cpm Nil (Ag, stimulation with adherent cells and antigen; Nil, stimulation with adherent cells and no antigen) shown in parentheses. 'Line was restimulated with 1 p-g/ml insulin. The sixfold stimulation with human insulin was significantly higher than Nil (P < .05, two-sample Student's t test). tLine was stimulated with 100 p-g/ml insulin. Pork and beef insulin stimulations were significantly higher (P < .001 and P < .02, respectively) than Nil. tLine from a diabetic patient was stimulated with 100 (JLg/ml insulin. Only beef insulin restimulation was significant compared with Nil (P < .01). §Lines were insulin specific, because they could not be restimulated with adherent cells and purified protein derivative (PPD), although fresh peripheral blood lymphocytes proliferated markedly (not shown).

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HYPOGLYCEMIA AND AUTO1MMUNITY SPECIFIC TO HUMAN INSULIN/I. SKLENAR AND ASSOCIATES

specific line from S.M. and two lines that also reacted with pork and beef insulins. Together with the PBL proliferation data this shows a cross-reactivity of most of S.M.'s T-lymphocytes with common antigenic determinants that are expressed on all three insulin species (23).

DISCUSSION

W

e describe an unusual patient (S.M.) with impaired glucose tolerance and postprandial hypoglycemia associated with abnormal humoral and cellular immunity to insulin. Her history did not reveal administration of exogenous insulin. Objective criteria, e.g., the failure of her serum to bind pork and beef 125 !-insulins, the presence of IgGs specific for human insulin, and the development of symptoms before human insulin became commercially available, strongly mitigate the possibility of exogenous immunization. The normal (nonsuppressed) level of C-peptide during spontaneous hypoglycemia also refutes the diagnosis of factitious hypoglycemia in this patient. However, in the presence of insulin antibodies the C-peptide level can be inappropriately high in autoimmune hypoglycemia (7) and factitious hypoglycemia (9). Therefore, it seems that C-peptide analysis cannot always reliably distinguish between factitious and autoimmune hypoglycemia. Total plasma insulin concentration of 0.43 nM = 65 pXJ/ ml (normal, 5-20 |xU/ml) was distinctly elevated during the episode of spontaneous hypoglycemia but was not excessively high, because it is frequently observed in factitious hypoglycemia (24). The possibility that the measured total insulin concentration was spuriously low due to the uniquely higher binding capacity of the autoantibody for human insulin than for the animal insulin tracer used in the insulin RIA cannot be excluded. The binding capacity of the insulin autoantibody estimated in the Scatchard plot with human insulin as ligand was very high (30,000 |xU/ml). A missed insulinoma in our patient was very unlikely, because no hypoglycemia developed during repeated fasts even after 72 h. Prolonged fasting was found to be the best provocative test for insulinoma (25). Careful dissection of excised pancreas and palpation of the remaining pancreas revealed no tumor. In addition, 5 yr after surgery the patient showed no progressive disease. However, insulinoma should always be considered a possibility in hypoglycemia because it can be associated with insulin auto immunity (26). The pancreatic insulin of our patient had a normal amino acid composition, as demonstrated by HPLC. However, certain drugs containing the sulfhydryl group (e.g., methimazole) may be associated with insulin autoimmunity, because they could render insulin immunogenic (7,27). This is of interest because our patient had Graves' disease and was treated with the sulfhydryl-containing drug carbimazole 7 yr before hypoglycemia developed. The combination of high total plasma insulin, high proinsulin, and high C-peptide levels after oral glucose suggests both increased insulin binding due to antibodies and inap-

propriately high endogenous insulin production after glucose stimulation. Benson et al. (7) suggested that insulin antibodies per se may stimulate insulin secretion. They observed pancreatic P-cell hyperplasia in a patient with recurrent hypoglycemia and insulin-binding antibodies; another patient showed markedly elevated plasma insulin and C-peptide levels during oral glucose tolerance testing, which were similar in our patient (7). In addition, human sera containing insulin antibodies have been reported to enhance glucose-stimulated insulin secretion from isolated rat pancreatic islets (28). Therefore, it is conceivable that the inappropriate suppression of endogenous insulin secretion during hypoglycemia in S.M. was due to such effect of insulin antibodies. C-peptide levels were abnormally elevated during the oral glucose tolerance test, but at hypoglycemia (240 min after oral glucose) they normalized; analogous C-peptide behavior occurred possibly during the episode of spontaneous postprandial hypoglycemia because it was in the normal range. Similar findings of C-peptide concentrations after an oral glucose tolerance test in a patient with insulin autoimmunity were reported by others (7). Release of insulin from the antibody depot, which is not subject to feedback regulation by glucose levels, may represent another mechanism for hypoglycemia (1-6) and could have also played a role in S.M. However, we were unable to find any change in the binding affinity or capacity of S.M. 's serum for human insulin during the course of oral glucose tolerance testing. Recently, it was suggested that, rather than sequester a hormone, binding proteins may augment its local availability in the tissues (29). The circulating helper-inducer T-lymphocytes abnormally reactive to stimulation with insulin may have been the cause of insulin autoimmunity in this patient. They might have provided a helper function for B-lymphocyte clones capable of autoantibody production (30-32). Our patient developed immune hypoglycemia in the context of polyendocrine autoimmunity, because she had already suffered from Graves' disease. Thyroid autoimmunity has been extensively studied as a model for autoimmune mechanisms (33,34). Clonal deletion of self-reactive B-lymphocytes, either absolute or functional in the absence of antigen-specific helper-inducer T-lymphocytes, seems to guarantee under normal circumstances that the immune system does not respond to self. A hypothesis based on animal data views autoimmunity as due to the appearance of autoreactive helper T-lymphocytes (possibly by somatic mutation), which then persist because of a defect in suppressor T-lymphocytes (34). The demonstration of such helper-inducer T-lymphocytes in our patient with a polyendocrine type of autoimmunity provides further support for this hypothesis. The antibody response of B-lymphocytes may be restricted when compared with the T-lymphocyte response in the same organism (35). Thus, it has been shown that BALB/c mice producing antibody specific either to pork or guinea pig insulin could, after priming with the latter, generate helper T-lymphocytes that cross-reacted with pork insulin despite major differences in amino acid composition. An analogous


157


situation is shown for S.M., whose B-lymphocytes produced only human insulin-specific antibody, but whose T-lympho' cytes were sensitized to epitopes shared by human, pork, and beef insulins. Similarly, in diabetics who were treated only with beef and pork insulins, lymphocyte proliferative r e ' sponses were induced with beef, pork, and human insulin species (23). This report emphasizes the importance of using a human insulin ligand when testing for autoantibodies to insulin. Autoantibodies specific for human insulin were first described in 1984 (36), before which insulin antibodies were believed to always be cross-reactive, although sometimes unequally, with the human, pork, and beef variants. T h e strong specificity of S.M.'s serum for human insulin was evident from adsorption studies. Note that porcine differs from human insulin by a single substitution out of 51 residues. A n ELISA modified to detect K- or \-light chains indicated that the insulin autoantibodies from S.M. were monotypic, type \ ; there was n o evidence for a myeloma as shown by repeatedly normal serum and urine immunoelectrophoresis. This suggests that her insulin autoantibodies were monoclonal and is supported by the findings of a single binding affinity by Scatchard analysis. Antibody responses to heterologous proteins are usually polyclonal, but it has been recognized that autoimmune responses show considerable epitope restriction and may be monoclonal, e.g., in Graves' disease (30,31). T h e role of insulin autoantibodies as serum markers for insulitis that causes diabetes mellitus has been recently discussed (37,38). S.M. was glucose intolerant in addition to suffering from hypoglycemia, but we do not know whether the intolerance was due to pancreatic damage or to a loss of insulin activity caused by insulin autoantibodies. In pancreatic sections obtained by surgery, n o insulitis was found. S.M.'s serum did, however, contain IgG islet cell antibodies both to fixed and to fresh-frozen pancreases. T h e findings in this patient are of clinical significance because the combination of hypoglycemia, elevated total insulin, and proinsulin in the absence of fasting hypoglycemia may suggest atypical insulinoma (39,40). Insulin autoimmunity should be suspected if this condition is associated with a marked difference between total and free plasma insulin with PEG extraction of the plasma.

Address correspondence and reprint requests to Dr. Ivo Sklenar, Institute for Microbiology and Hygiene, University of Basel, Petersplatz 10, CH-4003, Basel, Switzerland.

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