Anti-insulin receptor antibodies in human diabetes

Diabetes Research and Clinical Practice. I (1989) S59-S66

s59

Elsevier

DIABET

KO041

Anti-insulin receptor antibodies in human diabetes Ryushi Shimoyama, Yoko Fujita-Yamaguchi

and Giinther Boden

Research Division, Hokkaido Red Cross Blood Center, Supporo. Japun. Department of Moleculur Genetics, Beckmarl Research Itlsritute of’the City qf Hope. Duarte. CA, U.S.A. and General Clinical Research Center, Temple University Hospital, Philadelphia, PA. L:.S.A.

Key words: Anti-insulin assay

receptor

,antibodies;

Type B insulin resistance

with acanthosis

nigricans;

Insulin receptor

immune

precipitation

Summary Sera and immunoglobulin G from lo/104 diabetic patients (five with insulin-dependent, five with non-insulindependent diabetes) were found to contain antibodies that bound 1251-labelled purified human placental insulin receptors. Nine of these sera failed to inhibit insulin binding to cultured rat hepatocytes and did not stimulate glucose oxidation in rat adipocytes. Only one serum modestly inhibited insulin binding and stimulated glucose oxidation. These results suggest that sera from nine of these 104 diabetics contained a new type of anti-insulin receptor antibodies (AIRA) which bound to a locus different from the insulin binding site, and that only one of the 104 diabetic sera contained a low titer of conventional AIRA which could cause a clinical condition not distinguishable from ordinary non-insulin-dependent diabetes. -.

Introduction Anti-insulin receptor antibodies (AIRA) cause insulin resistance, non-ketotic diabetes and acanthosis nigricans [ 11. Recently, Maron et al. [2] reported

Address

for correspondence:

Division,

Hokkaido

Sapporo

060, Japan.

0168-8227/X9/$03.50

R. Shimoyama,

Red Cross Blood Center,

M.D., Research South 9 West 14,

10 1989 Elsevier Science Publishers

that 10 of 22 diabetic children had AIRA at the time of diagnosis. This suggested that AIRA may not be rare and may be pathogenetically important. We have measured AIRA with two different assays (binding inhibition and immune precipitation) in plasma from 5 1 insulin-dependent diabetics 53 non-insulin-dependent diabetics (IDDM). (NTDDM), four people with type B insulin resistance with acanthosis nigricans, 34 people with rheumatic diseases, eight with polycystic ovary (PCO) disease and 43 normal controls.

B.V. (Biomedical

Division)

S60

S61

Materials and methods Subjects

Some details of the subjects are shown in Table 1. Plasma was drawn after an overnight fast from the IDDM patients, in 20 of whom insulin therapy had not been started yet. Forty-one of the NIDDM patients were on insulin therapy, 9 of whom were insulin-resistant. Gamma-globulin

m

Gamma-globulin was extracted from some of the sera by the protein A sepharose method [3]. Primary culture

qf rat hepatocytes

Rat hepatocytes were obtained as previously described [4]. Briefly, hepatocytes were digested in situ by collagenase, centrifuged and plated in a flask at a density of 4.106/mm2 in minimal essential medium containing 10% neonatal calf serum.

100

10 UNLABELED

Fig. 1. Displacement

by unlabelled

10000

1000

INSULIN

ADDED

(ng)

insulin of 13’I-insulin

to 1251-insulin

receptors

(circles) or to unlabelled

tors (squares).

Shown are means of duplicate

bound

insulin recep-

determinations.

IR,

insulin receptor.

Insulin binding to hepatocytes

Cultured hepatocytes were preincubated with patients’ serum for 2 h. The medium was washed with KRB and incubated with a tracer amount of 1251insulin in the presence of native insulin. Iodination of human placental insulin receptor

The insulin receptor was purified from human placenta by the method of Fujita-Yamaguchi et al. [5]. Iodination of the purified insulin receptor was done by the Chloramine T method in Tris-HCl buffer [6]. The ‘251-insulin receptor was purified over Sephadex G-25-WGA column. As seen in Fig. 1, there were similar levels of ‘311-insulin binding to the labelled and unlabelled receptors and a similar displacement of ’ 3‘I-insulin by native insulin from the labelled and unlabelled receptors. Immune precipitation of the insulin receptor

Patient serum was incubated with a labelled insulin receptor preparation for 24 h and the precipitate was washed with buffer and counted in a gammacounter.

Hepatocyte insulin-binding-inhibition assay

Insulin binding to hepatocytes was examined as described elsewhere [7]. Briefly, hepatocytes in a monolayer were preincubated with sera from the various study groups for 2 h at 37°C and then washed twice with KRB buffer. After that, the cells were incubated in 2.5 ml KRB buffer containing 1% bovine serum albumin (BSA) ‘251-insulin with or without native insulin. Insulin binding in the presence of 10,000 ng of insulin was regarded as non-specific and subtracted from the total binding. Preparation of isolated adipocytes

Isolated adipocytes were prepared from the epididymal fat pads of ad-lib-fed male Sprague-Dawley rats by the method of Rodbell [8] as modified by Cushman [9]. Briefly, epididymal fat pads were minced and digested at 37°C with crude collagenase in KRB buffer, pH 7.4, containing 0.1% BSA. After 60 min of digestion the liberated cells were washed by centrifugation and resuspended in the same buffer.

S62 Glucose oxidation Isolated

hioassq~

rat adipocytes

$0 50

were incubated

for 2 h at

B

P”’

t

37°C in KRB buffer containing 0.1 mM glucose and 0.1 mCi/ml l-[‘4C]glucose in rubber-capped polyethylene bottles in a metabolic shaker bath. After that, 0.5 ml of 6 N H,SO, was added and the incubation

continued

generated paper,

for another

was trapped

in a No.

which had been soaked

hour.

The ‘“CO2

1 Whatman with hyamine

filter hy-

droxide and positioned in wells hung inside the incubation bottles. After 1 h of incubation the wells were cut off and submerged in ACS scintillation liquid and counted in an automatic scintillation counter. Analytical procedures Glucose was determined with a Beckman glucose analyzer. Free insulin was measured by radioimmunoassay (RIA) [IO]. Plasma anti-insulin antibodies were determined according to Gerbitz and Kemmler [I 11. Statistical evaluation was performed with the paired and unpaired

Student’s

IO *

IO q

SERUM

IO 1

IO 1

0

IO J

DlL"TlON

200

,NS"L,N

Fig. 2. Left panel: effects of serial dilutions

600 RECEPTOR

1000

"$8

ADDED

of four sera from

patients

with type B severe insulin resistance with acanthosis nigricdns. “51-labe11ed, human purified insulin receptors were

incubated

for 24 h at 4°C with serum.

tors were preincubated The number

of control

panel: displacement containing

sera is indicated

of ‘*51-insulin receptors

serum (from patient

insulin receptor

Bound

with rabbit anti-human

protein.

I. dilution

“‘l-insulin

recep-

gamma-globulin.

in parentheses.

Right

bound to an AIRA1:lOOO) by unlabelled

Shown are means of triplicate

determi-

nations.

f-tests.

Results

3.0 i 0.4%. The right panel of Fig. 2 shows the displacement of iZ51-insulin receptor binding to serum from patient 1 (dilution 1: 1000). Addition of unlabelled insulin receptor resulted in a progressive decrease of ‘251-insulin receptor binding from 28% to 2.4% with the addition of 1000 ng of receptor protein. Addition of unlabelled insulin or glucagon in doses ranging from 100 to 10,000 ng/ml did not

’ 25I-insulin receptor binding and displacement Nonspecific binding, i.e., radioactivity immunoprecipitated after the incubation of ‘251-labelled insulin receptor with buffer alone, was 4.1 rtO.3%. r2’linsulin receptor binding after incubation with con-

affect insulin

trol sera (dilution 1: 100) was 3.4* 0.3%. The intraand inter-assay coefficients were 0.07 and 0.12 respectively. There was no difference between “‘Iinsulin receptor binding by control sera and binding by the equivalent amounts of immunoglobulin G (IgG) extracted from these sera. ‘251-insuhn receptor binding increased in a concentration-dependent fashion after incubation with sera from four patients with type B severe insulin resistance. reaching 20. 31, 41 and 48% at serum dilutions of I: 100 for patient 2. B-IO, patient 1 and D.L., respectively (Fig. 2, left panel). In contrast, 12”I-insulin receptor binding after incubation with increasing amounts of normal (control) sera increased to a maximum of

Insulin receptor binding to .sera ,fiom diubetic, rlleumatic and PC0 putients Insulin receptor binding to sera (dilution 1: 100) from one of eight patients with PCO, one of 34 patients with rheumatic diseases, five of 51 patients with IDDM. five of 53 patients with NIDDM and all four patients with type B severe insulin resistance exceeded the normal range, defined as the mean + 3 SD of 43 normal control sera (Fig. 3). ‘251-insulin receptor binding by the insulin receptor immune precipitation assay (IRIP)-positive sera was concentration-dependent (Fig. 4) and was comparable with serum and IgG. As seen in Fig. 5 (right panel)

receptor

binding.

S63 I

100

.

.

. .

r=0.73

S: r = 0.98

Y IO

43

34:

‘8

(51

Fig. 5. Correlation

4,

(53

between

en AIRA-containing Fig. 3. ‘251-insulin

receptor

from normal

controls

cystic ovary

syndrome

binding

to sera (dilution

(NC. n = 43) and from patients (PCO.

n = 8). rheumatic

1:lOOO)

with poly-

disorders

(n =

n = 52).

non-

insulin-dependent 34) insulin-dependent diabetes

diabetes

(IDDM,

(NIDDM,

n= 53) and type B insulin

resistance

nigricans

indicate

with acanthosis the normal

range (mean

(n=4).

The horizontal

f 3 SD of normal

of IgG extracted shows

IO

20 %

iz51-insulin

sera (vertical

receptor

binding

axis) and equivalent

from these sera (horizontal

I:100 dilution.

20 %

the right panel

to sevamounts

axis). The left panel

I:1000 dilution

of serum

and IgG.

lines

controls).

insulin

receptor

immune

precipitation

of seven

IRIP-positive sera (dilution 1: 1000) and equivalent amounts of IgG prepared from these sera were highly correlated. Clinical characteristics of the 12 IRIP-positive patients are presented in Table 2.

Inhibition qf insulin binding Insulin binding levels to hepatocytes preincubated with sera (dilution 1:lOO) from all eight patients with PCO, all 34 patients with rheumatic disease, all 51 patients with IDDM and all 53 patients with NIDDM were within the normal range. Three of the four type B patients with insulin resistance had inibited insulin binding of 36, 29 and 5 1% of normal respectively, while patient 2’s serum was normal in this dilution. This latter finding indicated that the hepatocyte assay was less sensitive than the IRIP assay. At a lo-fold higher concentration all four sera from the type B patients were strongly inhibitory. Serum and IgG from one NIDDM patient (E.T.) significantly inhibited insulin binding while the other 11 IRIP-positive sera remained noninhibitory. SERUM

Fig. 4. Effects of serial dilutions

DILUTION

of the 12 IRIP-positive

sera and

I4 control sera on ‘251-insulin receptor binding. Because of their scarcity, some sera were not tested at higher dilutions.

Glucose oxidation Stimulation of glucose oxidation by 100 ng/ml of insulin was 380& 27% of buffer controls (Fig. 6).

2

71

M

F

F

W.H.

R.M.

R.C.

disease

70 63

F

31 45

M F

M.S.

AN, acanthosis

F

37

HT. hypertension;

insulin.

nigricans:

IRI. immunoreactive

LB.

PC0

E.M.

Rheumatic

A.R.

81

9

F

C.H.

F

40 16

F M

J.K.

K.W

IDDM

61 58

F

AIRA

15

22

75 24

16

10 25

4x

20

136

15

95

CpUiml)

IRI

___~~~

IR. insulin resistance

124/64

157173

196/95 165162

137/38

169182 160155

1681108

161173

178175

1571123

1651106

Height/Weight

(cm/kg)

(years)

WITH

Age

OF I2 PATIENTS

H.E.

Sex

CHARACTERISTICS

E.T.

NIDDM

Patients

CLINICAL

TABLE

Yes

No

No

I 25

Yes

32

33

35

45

0

40

x

87

40

0

0

200

(U/d) ~~~~~

requirement

Insulin

~~~~

37

16

30

No

Yes

Yes

Yes

Yes

3

I2

No ND

32

Yes

(years) ___

Duration of diabetes

Insulin antibodies

____~

~~

TSed. rate

Multiple

arthritis

thyroiditis

thyroiditis

thyroiditis

sclerosis

Rheumatoid

Hypoglycemia

Hashimoto’s

Hypopituitarism

Hashimoto’s

HashimotoS

AN. HT. cholesterol

+ Syph. serology

AN. IR

HT. Osteoarthritis

AN, IR, Vitiligo.

Comments

~_____

S65 assay. This assay measured the ability any type to bind ‘251-labelled, highly man

placental

insulin

receptors.

of AIRA of purified hu-

Its validity

was

supported by the demonstration that sera from four patients with type B insulin resistance, but not normal sera, increased ‘251-insulin receptor binding in a dose-dependent manner. Moreover, ‘251-insulin receptors labelled

bound insulin

to AIRA could be displaced receptor

protein.

possible after full purification

Fig 6. Etfects of sera (dilution (hatched

column),

three

columns),

itory AIRA

sented as percent 2-h incubation

with binding-inhibitory

and nine patients

(open columns)

case from isolated

1:50) from eight normal

patients

rat adipocytes. of maximal

Data

(mean

AIRA

from l-[14C]gluf

SD) are pre-

insulin effect (I 00 ng/ml) during

at 37°C. Sera from D.L., patient

ulated significantly

controls

with non-binding-inhib-

on CO production

more CO production

a

I and E.T. stim-

than did those of con-

trols.

Stimulation of glucose oxidation by control sera (dilution 1:50) was 14.2 f 11.9% of the effect of 100 ng/ml insulin. Nine of the 10 IRIP-positive sera did not stimulate glucose oxidation significantly more than did control sera. In contrast, stimulation by serum E.T., which also inhibited insulin binding to hepatocytes, significantly elevated it comparably to the two type B sera tested. AZA and insulin concentrations Five of the 10 TRIP-positive sera tested contained AIA; they bound more insulin than did the 25 control sera. Insulin binding, however, did not correlate with insulin receptor binding. Six of the IRIPpositive sera had elevated serum insulin concentrations. Serum insulin did not correlate with insulin receptor binding.

Discussion To test for AIRA, which do not inhibit insulin binding, we have developed a new immune precipitation

of the insulin receptor

and its direct labelling. Similar immune precipitation assays have been performed by Harrison et al. [12] and Tushima et al. [13], but they used 1251insulin-labelled or insulin-cross-linked crude pla-

(8) (6) (3) (4) (3) (4) (2) (4) (1) (2) (1) (4) (1)

(black

by un-

This assay became

cental membrane fractions. Using this new IRIP assay, we found that 12 sera and their IgGs were positive for the presence of AIRA, but that only one serum was positive for the insulin binding inhibition assay in cultured rat hepatocytes and human placental membranes. On the other hand, this serum was not positive for inhibition of ‘251-IgF1 binding to its receptor. These results suggested that 11 of the 12 IRIPpositive sera contained antibodies that bound to the insulin receptor but at a locus different from the insulin binding site. Evidence for the presence of different species of AIRA has been provided by De Pirro et al. [14], using affinity column chromatography of type B serum, but the sole presence of this kind of AIRA in human serum has not been shown so far. However, the functional significance of these antibodies remains uncertain. A second objective of this study was to determine the prevalence of conventional binding-inhibitory AIRA in the diabetic population. We were able to find such antibodies in only one serum of 104 diabetic sera. Ludwig et al. [15] found that three of 29 newly diagnosed diabetic children had low titers of insulin binding-inhibitory AIRA. Maron et al. [2] reported AIRA of the IgM class in 10 of 22 young, untreated IDDM patients. But they defined AIRA by the serum’s ability to stimulate lipogenesis in rat adipocytes. Since many substances other than AIRA can exert this insulin-like activity, it is not certain that their sera contained AIRA. In conclusion, using a newly developed immune

S66 precipitation AIRA

assay. we found

which bound

insulin binding tively common tional

insulin

that a new type of

to a locus different

from the

site in the insulin receptor was relain diabetic sera, but that convenbinding-inhibitory

AIRA

were rare.

7 Shimoyama.

R.. Shelmet,

J.J.. Savage,

G. (1986) Effects of anti-insulin on downregulation

and turnover

tured hepatocytes.

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antibodies

(AIRA)

of insulin receptors

on cul-

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hormones

C.R. Jr. and Boden,

receptor

of isolated fat cells. Effects of

metabolism

and

lipolysis.

J. Biol.

Chem. 239, 3755380. 9 Cushman.

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