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The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 1
Printed in U.S.A.
Insulin and Glucagon Secretion in Patients with Slowly
Progressing Autoimmune Diabetes (LADA)*
ÅSALINDA CARLSSON, GÖRAN SUNDKVIST, LEIF GROOP,
TIINAMAIJA TUOMI
AND
Department of Endocrinology, University of Lund, S-205 02 Malmo, Sweden (Å.L.C., G.S., L.G., T.T.)
and the Department of Internal Medicine, Helsinki University Central Hospital (T.T.),
Helsinki, Finland
insulin concentrations (P , 0.04). In contrast, b-cell sensitivity to
glucose was unaltered in LADA and type 2 diabetes. The glucagon
concentrations were elevated in both LADA and type 2 diabetic patients compared with healthy control subjects (P , 0.02), but did not
differ between the diabetic groups. In conclusion, patients with LADA
share insulin resistance with type 2 diabetic patients, but display a
more severe defect in maximally stimulated b-cell capacity than patients with type 2 diabetes. (J Clin Endocrinol Metab 85: 76 – 80, 2000)
ABSTRACT
To metabolically characterize patients with slowly progressing autoimmune diabetes (LADA) of short duration we measured insulin, C
peptide, and glucagon responses to glucose and arginine at three blood
glucose levels (fasting and 14 and 28 mmol/L) in 11 patients with
LADA, 11 patients with type 2 diabetes, and 14 healthy control subjects matched for age and body mass index. The acute insulin response
to arginine was impaired in LADA vs. type 2 diabetes at all glucose
levels, with the greatest impairment in the maximally stimulated
A
BOUT 10% of patients diagnosed with type 2 diabetes
have circulating autoantibodies to either islet cell cytoplasmic antigens (1–3) or, more frequently, glutamic acid
decarboxylase (GADab) (4 –9). This subgroup, also referred
to as latent autoimmune diabetes in adults (LADA) (5, 9), has
been included in the new WHO proposal for classification of
diabetes as a slowly progressing form of type 1 diabetes (10).
Obviously LADA shares features with both type 1 and type
2 diabetes. Although insulin secretion is better preserved in
the slowly progressing than in the rapidly progressing form
of autoimmune diabetes (9, 11), insulin secretion tends to
deteriorate with time in LADA patients (7–9, 12). Assessments of insulin secretion in the earlier studies was based
upon crude measurements of b-cell function, such as fasting
or glucagon-stimulated C peptide concentrations (3, 5, 7, 13,
14) or the HOMA model of b-cell function (8). These tests
would hardly stress the b-cells to their limits to allow detection of more subtle defects in b-cell capacity. Also, in most
studies the patients have had a relatively long duration of the
disease, which makes it difficult to discern between primary
(autoimmune) and secondary (chronic hyperglycemia) defects in insulin secretion. Glucagon levels and responses differ between type 1 and type 2 diabetes during the course of
the disease (15–19). No information is available on glucagon
secretion in patients with LADA. The aim of this study was
to compare insulin and glucagon secretion and insulin sensitivity between LADA and type 2 diabetic patients of short
duration and healthy control subjects.
Subjects and Methods
Subjects
Between 1994 –1996, 573 consecutive patients diagnosed with type 2
diabetes in Malmö, Sweden were tested for GADab. Of them, 49 (8.5%)
were GADab positive (GADab1). Eleven of these GADab1 patients met
the criteria (.30 –70 yr of age, no insulin treatment during the first 12
months) and accepted to participate in an iv glucose-arginine test. They
were matched for age, sex, and body mass index (BMI) with 11 GADabnegative (GADab2) type 2 diabetic patients diagnosed during the same
period. Fourteen nondiabetic individuals without first degree family
history of diabetes served as control subjects (Table 1). Informed consent
was obtained from all participants. The local ethics committee at Lund
University approved the study.
Glucose-arginine test
Insulin secretion was measured during iv stimulation with glucose
and arginine after an overnight fast according to the method described
by Ward et al. (20) (Fig. 1). Blood samples were drawn for measurement
of glucose, insulin, and C peptide (the latter only for diabetic patients)
5 and 2 min before as well as 2, 3, 4, and 5 min after an iv injection of
5 g arginine hydrochloride. Glucagon was measured only at 25 and 22
min. This procedure was carried out in the basal state and after the blood
glucose concentration had been raised to 14 and 28 mmol/L [mean
coefficient of variation (CV), 8.1% and 7.0%, respectively) by a variable
iv infusion of 20% glucose. Blood glucose was measured every 5 min to
maintain the desired blood glucose concentration. A 2.5-h resting period
was allowed before the blood glucose was raised to 28 mmol/L to avoid
the priming effect of hyperglycemia. After new baseline samples, the
glucose infusion was restarted, and blood glucose was raised to 28
mmol/L over 25–30 min. The insulin response to glucose at fasting blood
glucose and at blood glucose levels of 14 and 28 mmol/L was calculated
as the mean of the insulin levels at 22 and 25 min. The acute insulin
response to arginine (AIRarg) was calculated as the mean of the 2–5 min
values after subtraction of the mean of the prestimulus values. The C
peptide response to glucose, acute C peptide response (ACRarg) to
Received May 25, 1999. Revision received August 31, 1999. Accepted
September 9, 1999.
Address all correspondence and requests for reprints to: Tiinamaija
Tuomi, M.D., Wallenberg Laboratory, Department of Endocrinology,
University of Lund, S-205 02 Malmo, Sweden. E-mail: tiinamaija.tuomi@
endo.mas.lu.se.
* This work was supported by grants from the Påhlsson Foundation,
the Medical Faculty of the Lund University, the Malmö University
Hospital, the Swedish Society of Medicine, the Crafoord Foundation, the
Swedish Medical Doctors Association, and the Malmö Diabetes
Association.
76
INSULIN AND GLUCAGON SECRETION IN LADA
77
FIG. 1. Serum insulin concentrations (y-axis) during the glucose-arginine test in patients with LADA (F; n 5 11) or type 2 diabetes (E; n 5
11) and in nondiabetic control subjects (Œ; n 5 14). The insulin concentrations are given at three blood glucose levels (fasting, 14 mmol/L, and
28 mmol/L) 5 and 2 min before as well as 2, 3, 4, and 5 min after an injection of arginine (marked with arrows).
arginine, and glucagon response to glucose (GRgluc) were calculated in
the same manner.
The AIRarg at a blood glucose level exceeding 25 mmol/L is considered to represent the maximal insulin secretory capacity (20). The
glucose potentiation of b-cells was estimated as the slope between
AIRarg at fasting and at a blood glucose level of 14 mmol/L (slopeAIR
5 DAIRarg/Dglucose) (21). From this equation, the b-cell sensitivity to
glucose was calculated as the blood glucose level at which half-maximal
AIR to arginine occurred (20).
Laboratory methods
Serum insulin concentrations were measured using a double antibody enzyme-linked immunosorbent assay (DAKO Corp., Cambridgeshire, UK) with an interassay CV of 8.9%. Serum C peptide concentrations were measured by RIA (Linco Research, Inc., St. Charles,
MO) with an interassay CV of 9.8%. Plasma glucagon concentrations
were measured by RIA (Linco Research, Inc.). In the assay, pancreatic
glucagon had a cross-reactivity with enteric glucagon of less than 0.1%
and an intraassay CV of 3.6%. Blood glucose was measured in duplicate
using the glucose oxidase method. GAD antibodies were measured from
frozen serum samples by a radioimmunoprecipitation assay employing
recombinant human [35S]GAD65 produced by in vitro transcription/
translation as described previously (9). At the Combined Autoantibody
Workshop, the specificity of the assay was 99%, and the sensitivity was
75% (22).
Statistical analysis
Statistical analyses were performed using the BMDP new system,
version 1.12, for Windows (BMDP Statistical Software, Inc., Los Angeles,
CA). Data are given as the mean 6 sd or as the median (75–25% interquartile range) unless indicated otherwise. The statistical significance of
the difference between groups was tested using the Mann-Whitney
individual rank sum test.
Results
Patients with LADA and type 2 diabetes were matched
with respect to sex, BMI, and age at diagnosis of diabetes
(Table 1). The LADA patients had a slightly shorter duration of diabetes than the type 2 diabetic patients (1.7 6
1.2 vs. 3.2 6 1.2 yr; P 5 0.01). Insulin resistance according
to the HOMA model (23) did not significantly differ between LADA and type 2 diabetic patients [median (75–25%
interquartile range), 2.1 (3.9) vs. 3.2 (3.5)]. However, both
diabetic groups differed significantly from the control subjects [median (75–25% interquartile range), 0.4 (1.3); P ,
0.03; Table 1].
Insulin and C peptide responses
Both diabetic groups showed significantly lower insulin
responses to glucose and glucose plus arginine compared
with the control subjects of similar age and BMI (Fig. 1).
Insulin secretion stimulated by glucose was decreased in
LADA compared with type 2 diabetic patients at 28 mmol/L
(12.2 6 9.0 vs. 24.3 6 25.8 mU/L; P 5 0.04; Fig. 2A). The acute
insulin response to glucose and arginine was significantly
lower in LADA compared with type 2 diabetic patients at all
blood glucose concentrations [AIRargf, 23.7 6 19.6 vs. 37.2 6
22.0 mU/L (P 5 0.03); AIRarg14, 28.6 6 17.2 vs. 50.8 6 29.0
mU/L (P 5 0.04); AIRarg28, 42.1 6 36.1 vs. 80.9 6 62.6 mU/L
(P 5 0.04); Fig. 2B]. Similarly, the C peptide responses to
glucose and arginine were lower in LADA than in type 2
diabetic patients (Fig. 2, A and B).
78
JCE & M • 2000
Vol 85 • No 1
CARLSSON ET AL.
TABLE 1. Clinical characteristics of the subjects
n (m/f)
Age (yr)
Age at diagnosis (yr)
Duration (yr)
BMI (kg/m2)
fB-glucose (mmol/L)
HbA1c (%)
fS-C-peptide (nmol/L)
Insulin resistance (HOMA)
LADA
Type 2 diabetes
Control subjects
8/3
54.5 6 11.2
52.9 6 10.6
1.7 6 1.2a
27.3 6 4.1
9.6 6 4.3
7.6 6 2.3
0.9 6 0.4
3.5 6 4.0
8/3
57.0 6 9.0
53.3 6 9.4
3.2 6 1.2
26.2 6 2.7
8.6 6 2.2
6.3 6 1.0
1.1 6 0.5
3.7 6 3.6
4/10
54.4 6 10.7
N/A
N/A
26.2 6 5.0
5.0 6 0.5
4.6 6 0.4
ND
0.99 6 1.4b
Data are shown as mean 6 SD or median (75–25% interquartile range).
N/A, not applicable; ND, not done.
a
P 5 0.01 vs. type 2 diabetic patients.
b
P , 0.03 vs. both LADA and type 2 diabetic patients.
FIG. 3. Glucagon response to iv glucose at three blood glucose
levels (fasting, 14 mmol/L, and 28 mmol/L) in patients with LADA
(F; n 5 11) or type 2 diabetes (E; n 5 11) and in nondiabetic control
subjects (Œ; n 5 14). Data are shown as the mean 6 SEM. *, P , 0.02;
**, P , 0.007; ***, P , 0.001 (LADA and type 2 diabetes vs. control
subjects).
There was no significant difference in the glucose potentiation of b-cell function between LADA and type 2 diabetes,
whereas both diabetic groups differed significantly from the
control subjects [median (75–25% interquartile range):
slopeAIR, 1.3 (1.6) and 2.1 (3.3) vs. 5.4 (7.3); P , 0.0002]. b-Cell
sensitivity to glucose was unaffected in the two diabetic
groups [median (75–25% interquartile range): LADA vs. type
2 diabetes, 8.6 (12.1) vs. 10.7 (13.5) mmol/L] and was indistinguishable from that in control subjects [8.1 (3.9) mmol/L].
FIG. 2. Serum insulin and C peptide response to iv glucose (A) and
acute insulin or C peptide responses (AIRarg, ACRarg) to glucose and
arginine (B) at three blood glucose levels (fasting, 14 mmol/L, and 28
mmol/L) in patients with LADA (F; n 5 11) or type 2 diabetes (E; n 5
11) and in nondiabetic control subjects (Œ; n 5 14). AIR and ACR were
calculated as the mean of the 2–5 min values after subtraction of the
mean of the prestimulus values. Data are shown as the mean 6 SEM.
Note that the y-axis in B has a logarithmic scale. *, P 5 0.05; **, P 5
0.04; ***, P 5 0.03 (LADA vs. type 2 diabetes).
Glucagon response
The glucagon concentration was decreased by increasing
the glucose concentration in the control subjects (Fig. 3). In
the diabetic groups the glucagon concentration was elevated
and less suppressed by glucose compared with that in the
control subjects [LADA and type 2 diabetic patients vs. con-
INSULIN AND GLUCAGON SECRETION IN LADA
trol subjects: GRglucf, 64.4 6 18.2 and 68.7 6 30.8 vs. 44.8 6
12.6 ng/L (P # 0.02); GRgluc14, 56.4 6 19.1 and 60.1 6 24.1
vs. 31.9 6 9.2 ng/L (P # 0.001); GRgluc28, 39.2 6 12.0 and
42.9 6 19.5 vs. 24.6 6 7.6 ng/L (P # 0.007)]. No significant
difference was seen between LADA and type 2 diabetic patients with respect to the glucagon concentration (Fig. 3).
Discussion
The present study provided novel information on the
pancreatic defects in patients with LADA, which can be
used to distinguish them from patients with classical type
2 diabetes as well as from patients with classical type 1
diabetes. The tools used allowed careful quantification of
b-cell capacity and sensitivity (for both insulin and glucagon secretion). Although patients with LADA shared
insulin resistance and elevated glucagon levels with patients with type 2 diabetes, they exhibited a much more
severe defect in insulin secretion. Prospective studies have
previously shown that GADab1 patients deteriorate in
their insulin secretion over time and that they are more
likely to require insulin therapy than GADab2 patients (7,
8, 12). Although they in this respect resemble patients with
type 1 diabetes, we have shown that the LADA patients
have higher C peptide levels even after 10 yr of diabetes
than patients with adult-onset type 1 diabetes (9). Also, in
the present study the LADA patients did not require insulin treatment and had a mean fasting C peptide concentration of 0.64 6 0.32 nmol/L after a disease duration
of 1.7 6 1.2 yr, separating them from patients that could
be classified as having type 1 diabetes. However, it has not
been known whether a difference in insulin secretory capacity between LADA and type 2 diabetes can be detected
at the time of diagnosis or soon thereafter. We showed in
patients with short term diabetes that despite similar fasting C peptide concentrations, a marked difference in b-cell
function between LADA and type 2 diabetes could be
observed at a high glucose concentration (28 mmol/L)
with and without stimulation with arginine.
Some important conclusions can be drawn from these
findings. The b-cell defect characteristic of LADA is not
restricted to stimulation with glucose, suggesting that it is
not due to defects in glucose metabolism in the b-cell. The
impaired insulin secretion after stimulation with arginine at
glucose concentrations of 14 and 28 mmol/L, rather, suggests
a reduction of the maximal b-cell capacity (20) consistent
with an irreversible autoimmune destruction of the b-cells.
The defect in insulin secretion is hardly a consequence of
chronic hyperglycemia, as the two patient groups had similar
glycemic control. Neither could it be a consequence of better
insulin sensitivity, as insulin secretion adjusted for insulin
sensitivity (HOMA) was impaired in both LADA and type 2
diabetic patients.
Despite an accelerated deterioration of b-cell function,
the LADA patients share many features with common type
2 diabetes, e.g. elevated glucagon levels and insulin resistance. The elevated glucagon concentrations may have
several metabolic consequences. First, glucagon stimulates insulin secretion, and the elevated glucagon concentrations may serve to maintain insulin secretion and ex-
79
plain why LADA patients rarely develop total b-cell
dysfunction (15–19). Glucagon is also a potent stimulator
of gluconeogenesis and glycogen breakdown, resulting in
increased hepatic glucose output. Although we did not
measure hepatic glucose production in this study, we have
previously shown that patients with LADA have an enhanced basal rate of hepatic glucose production, which
cannot be suppressed by insulin (11). This study provides
a potential explanation for the hepatic insulin resistance,
i.e. elevated glucagon levels.
In conclusion, metabolically LADA shares features with
both type 1 and type 2 diabetes. With the former, LADA
patients share a severe and progressing defect in b-cell function. With the latter, they share insulin resistance and elevated glucagon levels. These data further emphasize the
need to consider LADA as a diabetic subgroup distinct from
both type 1 and type 2 diabetes.
Acknowledgments
Gertrud Ahlqvist, Britt Bruveris-Svenburg, Marianne Lundberg, and
Christina Rosborn are acknowledged for skillful technical assistance,
and Dr. Hillevi Larsson for help with recruiting the nondiabetic control
subjects.
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