Original Article

doi: 10.1111/joim.12116

Assessment of beta-cell function in young patients with type

2 diabetes: arginine-stimulated insulin secretion may reflect
beta-cell reserve
M. Sjostrand1, K. Carlson1, H. J. Arnqvist2, S. Gudbjornsdottir3, M. Landin-Olsson4, S. Lindmark5, L. Nystrom5,
M. K. Svensson3, J. W. Eriksson1,3 & J. Bolinder6
From the 1AstraZeneca R&D, M
olndal; 2Department of Clinical and Experimental Medicine, Link
oping University, Link
oping; 3Sahlgrenska
Academy Hospital, Gothenburg; 4Department of Endocrinology and Diabetology, Lund University Hospital, Lund; 5Department of Public Health
and Clinical Medicine, Ume
a University, Ume
a ; and 6Department of Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet,
Stockholm, Sweden

Abstract. Sj
ostrand M, Carlson K, Arnqvist HJ,
Gudbj
ornsdottir S, Landin-Olsson M, Lindmark S,
Nystr
om L, Svensson MK, Eriksson JW, Bolinder J
(AstraZeneca R&D, M
olndal; Link
oping University,
Link
oping;
Sahlgrenska
Academy
Hospital,
Gothenburg; Lund University Hospital, Lund; Ume
a
University, Ume
a; Department of Medicine, Karolinska
University Hospital Huddinge, Karolinska Institutet,
Stockholm, Sweden). Assessment of beta-cell function
in young patients with type 2 diabetes: argininestimulated insulin secretion may reflect beta-cell
reserve. J Intern Med 2014; 275: 3948.
Objective. Simple methods for the evaluation of
dynamic b-cell function in epidemiological and
clinical studies of patients with type 2 diabetes
(T2D) are needed. The aim of this study was to
evaluate the dynamic beta-cell function in young
patients with T2D with different disease durations
and treatments.
Methods. Overall, 54 subjects with T2D from the
Diabetes Incidence Study in Sweden (DISS) and 23
healthy control participants were included in this
cross-sectional study. Beta-cell function was
assessed by intravenous (i.v.) administration of
arginine followed by i.v. glucose. The acute insulin

Introduction
Type 2 diabetes mellitus (T2D) is a progressive
disease, which becomes manifest when endogenous insulin secretion is no longer sufficient to
compensate for insulin resistance. b-Cell failure
then gradually progresses in a majority of patients
[1] who eventually become insulin dependent. Even

and C-peptide responses to arginine (AIRarg and

Ac-pepRarg, respectively) and to glucose (AIRglu and
Ac-pepRglu, respectively) were estimated. Homeostasis
model assessment of b-cell function (HOMA-b) and Cpeptide assessments were also used for comparisons
between patients with T2D and control participants.
Results. AIRarg and Ac-pepRarg, but not AIRglu and
Ac-pepRglu, could differentiate between patients
with different disease durations. AIRglu values were
89% (P < 0.001) lower and AIRarg values were 29%
(P < 0.01) lower in patients with T2D compared
with control participants. HOMA-b and fasting
plasma C-peptide levels did not differ between the
T2D and control groups.
Conclusion. In young patients with T2D, the insulin
secretory response to i.v. glucose is markedly
attenuated, whereas i.v. arginine-stimulated insulin release is better preserved and can distinguish
between patients with different disease duration
and antidiabetic therapies. This suggests that the
i.v. arginine stimulation test may provide an estimate of functional beta-cell reserve.
Keywords: type 2 diabetes, b-cell function.

though insulin secretion generally decreases over

time in patients with T2D, the pattern and speed of
decline differ considerably amongst individuals [2].
Up until now, no pharmacological agent that prevents or restores beta-cell dysfunction in humans
has been found. Peroxisome proliferator-activated
receptor (PPAR) agonists, glucagon-like peptide-1
(GLP-1) analogues and DPP-IV (dipeptidyl peptidase

2013 The Association for the Publication of the Journal of Internal Medicine

39

M. Sj
ostrand et al.

IV) inhibitors may have such effects [36], but this

remains to be further demonstrated.
There is a need to establish methods that are
appropriate for an outpatient setting to evaluate
the long-term course of functional b-cell mass in
clinical and epidemiological studies. Homeostasis
model assessment of b-cell function (HOMA-b) is
often used to estimate b-cell function, but the
method is limited because it is based on fasting
glucose and insulin levels, only measures b-cell
function under fasting nondynamic conditions and
has not been validated in patients with T2D treated
with insulin [7].
The standardized glucose-potentiated arginineinduced insulin secretion method has been used
for more than 25 years to measure b-cell function
in T2D and is often considered to be the gold
standard measure [8]. However, a simpler, less
labour-intensive method is needed for use in large
studies and clinical trials. This standardized
method has been shown to correlate with a simpler
test using an intravenous (i.v.) bolus of glucose or
arginine in normoglycaemic healthy participants
[9] and in patients who have undergone pancreas
transplantation [10]; however, to our knowledge,
this methodology has not been tested in patients
with T2D.
Therefore, the aim of this study was to evaluate
dynamic b-cell function in young patients with T2D
with different disease durations and treatments
and in healthy control participants using i.v.
arginine and glucose stimulation. b-Cell function
was also compared between patients and healthy
participants using the HOMA-b index, proinsulin/
insulin ratio and measurement of fasting plasma
C-peptide levels.
Methods
Study design
Overall, 54 subjects with T2D (diabetes duration 2
10 years) and 23 healthy control participants were
enrolled in this study D0280M00003. Patients
were recruited from the national Diabetes Incidence Study in Sweden (DISS) register, which
covers incident cases of diabetes in individuals
between 15 and 34 years of age and collects data in
collaboration with departments of internal medicine, endocrinology and paediatrics in Sweden. The
classification into type 1, type 2 and unclassified
diabetes in the DISS register is based on the
40

2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

The arginine stimulation test in young patients with T2D

treating physicians clinical diagnosis, according

to criteria of the World Health Organization [11]. In
this study, patients with T2D were recruited at five
university hospitals: Gothenburg, Ume
a, Link
oping, Lund and Huddinge, and the control group of
23 healthy participants were recruited at the
Gothenburg hospital. All patients were recruited
from a cohort of individuals in the DISS register diagnosed with diabetes between 1998 and
2006 (n = 397). All participants (male or female)
were invited to take part in the study and were
eligible if they met the following criteria: (i) aged
1534 years at the time of diabetes diagnosis and
inclusion in the DISS register; (ii) classified as
having T2D by the reporting physician; (iii) plasma
samples negative for islet-cell antibodies at or
within 34 months of diagnosis; (iv) diabetes
duration of 210 years; and (v) HbA1c <10%
(Swedish Mono-S standard; normal reference 3.0
5.3%). In total, 66 subjects with T2D were enrolled,
and 54 subjects were included in all analyses and
safety. The main reason for exclusion of enrolled
patients was unacceptably high plasma glucose
levels before the dynamic i.v. glucose and arginine
tests.
Male and female healthy (as judged by the investigator) control participants between 25 and 50 years
of age were recruited via advertisement and
included if they had a body mass index (BMI) of
1940 kg m 2 and did not have diabetes, impaired
fasting glucose or impaired glucose tolerance.
All participants gave written informed consent
prior to enrolment, and the study protocols were
approved by the ethics committee in Gothenburg.
The subjects with T2D were treated with diet
and exercise alone n = 10, oral hypoglycaemic
agents (OHAs) (metformin n = 33, thiazolidinediones n = 3, sulphonylurea n = 2, alpha-glucosidase
inhibitors n = 1) and insulin treatment n = 22 (10
treated with insulin only and 12 with insulin in
combination with OHA). Fig. 1 shows the distributions of treatment in patients included in
the DISS register <5 years, 5<8 years and 8
10 years, respectively, before the start of the
study.
At the enrolment visit (24 weeks before the
investigational day), all participants underwent
an examination including recording of demographic characteristics (sex and age), medical
history, family history of diabetes, current medi-

M. Sj
ostrand et al.

The arginine stimulation test in young patients with T2D

Included in DISS <5 years

n = 11

Included in DISS 5 to <8 years

n = 20

Included in DISS 8-10 years

n = 23

Fig. 1 Treatments in patients included in the DISS register <5, 5 to <8 and 810 years before the start of the study; OHA,
oral hypoglycaemic agents.

cation, weight and height (to calculate BMI), waist

and hip circumference, physical examination,
blood pressure, pulse and a laboratory screen.
In addition, in the control group, an oral glucose
tolerance test (OGTT) was performed to exclude
diabetes or impaired fasting glucose/glucose
intolerance. Patients with T2D and healthy control participants were matched for age, sex and
BMI.
On the investigational day, subjects who fulfilled
all the eligibility criteria for inclusion in the study
underwent testing in the morning after fasting
overnight. No medication, including insulin, was
taken on the morning of the investigational day. A
venous cannula was inserted into one arm for
blood sampling. Arginine and glucose (i.v.) for the
b-cell function test were administered in the other
arm. Blood samples were collected before the start
of the b-cell function test for analysis of fasting
plasma biomarkers including glucose, insulin,
proinsulin, C-peptide and HbA1c.

In addition, we obtained nonfasting plasma samples that had been collected close to diagnosis of
diabetes and inclusion in the DISS register and
stored at 80 C. These samples were analysed for
insulin, proinsulin and C-peptide levels at AstraZeneca R&D (M
olndal, Sweden).
Dynamic testing of beta-cell function
The i.v. b-cell function test was performed on the
investigational day according to the method of
Robertson [10]. Subjects with T2D were asked
not to take their usual pharmacological treatment
(including insulin) on the morning of the study day.
Before the test, glucose levels were measured to
ensure that they were between 4 and 12 mmol L 1.
A predose blood sample was collected immediately
before the start of the test. Arginine-induced insulin secretion was assessed by injecting an i.v. bolus
of 5 g arginine at time 0, and samples were drawn
2, 3, 4, 5, 7, 10, 25 and 30 min following the
injection. Immediately thereafter, glucose-induced
2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

41

M. Sj
ostrand et al.

insulin secretion was assessed by injecting an i.v.

bolus of 0.3 g kg 1 glucose, and samples were
collected at 3, 4, 5, 7, 10, 15, 20, 25, 30, 60, 115
and 120 min.
The acute insulin response to arginine (AIRarg) was
calculated as the mean of the three highest plasma
insulin levels obtained within 5 min after the
arginine bolus minus the prestimulus plasma
insulin level. The acute insulin response to glucose
(AIRglu) was calculated as the mean of the plasma
insulin levels obtained 3, 4 and 5 min after glucose
injection minus the prestimulus level. In addition,
the acute C-peptide response to glucose (Ac-pepRglu)
and the acute C-peptide response to arginine
(Ac-pepRarg) were calculated as described for insulin.
HOMA was calculated using the formula HOMAIR = (FPI 9 FPG)/22.5 and HOMA-b = (20 9 FPI)/
(FPG- 3.5) for insulin resistance and b-cell function, respectively, where FPI is fasting plasma
insulin concentration(mU L 1) and FPG is fasting
plasma glucose (mmol L 1) .
Analytical methods
Glucose concentrations in plasma were analysed
using a Cobas Mira Plus analyser (Hoffman-La
Roche, Basel, Switzerland) and an enzymatic (hexokinase) colorimetric method (ABX Pentra, Montpellier, France). HbA1c was assessed by highperformance liquid chromatography using Mono-S
ion-exchange chromatography (Pharmacia-Amersham Bioscience, Uppsala, Sweden). The reference
interval was 3.0% to 5.3%. Plasma concentrations of
insulin and C-peptide were measured using commercial radioimmunoassays (Millipore, St. Charles,
MO, USA). Proinsulin levels were measured using a
commercial enzyme-linked immunosorbent assay
(Mercodia, Uppsala Sweden). The total (intra- and
interassay) coefficient of variation was <3% for
glucose, <15% for insulin and <8% for C-peptide.
Cross-reactivity for the C-peptide and insulin
assays was <0.2% and <4% for human proinsulin
(HPI), respectively. Cross-reactivity of the HPI assay
was 84% for HPI Des (6465), 90% for HPI Split
(6566), 95% for HPI Des (3132) and 90% for HPI
Split (3233), respectively, and <0.03% and
<0.006% for insulin and for C-peptide, respectively.
Statistical analysis
Data are presented descriptively as means  SD.
Comparisons between groups at baseline were
42

2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

The arginine stimulation test in young patients with T2D

made using a two-sample t-test, and comparisons

between different lengths of diabetes duration and
different treatment groups were made using the
KruskalWallis test and subsequent post hoc analysis with the Dunns test. An exact Wilcoxon ranksum test was used for comparisons between
healthy control participants and patients with
T2D. Relationships were explored by Pearsons
correlation including confidence intervals based
on Fishers z-transformation.
Results
Clinical characteristics
Subjects with T2D were slightly younger than those
in the control group (34.6  5.4 vs. 39.6 
5.8 years); the two groups were well matched
for sex (male/female: 33/21 vs. 12/11), BMI
(33.2  5.7 vs. 32.6  4.5 kg m 2) and waist circumference (110  14.0 vs. 111  12.9 cm).
HbA1c was 6.2  1.3% (54 mmol mol 1) and 4.1 
0.3% (32 mmol mol 1) in the T2D and control
groups, respectively (P < 0.001). Clinical characteristics in subgroups based on diabetes duration
and antidiabetic treatment are shown in Tables S1
and S2.
Insulin and C-peptide responses after i.v. glucose and arginine
Fasting pretest glucose concentrations were
7.3  1.8 and 5.5  0.9 mmol L 1 in the T2D
and control groups, respectively (P < 0.001). Mean
fasting plasma insulin was almost twofold higher
in the T2D patient cohort than in the control group
(217  161 vs. 119  77 pmol L 1, P < 0.01). The
AIRarg and Ac-pepRarg values were 29% lower
(P < 0.01) and AIRglu and Ac-pepRglu values were
89% lower (P < 0.001) in patients with T2D compared with control participants (Table 1 and
Fig. 2a).
The sensitivity of the b-cells to respond to changes
in glucose (the glucose sensitivity) was also estimated by dividing the insulin responses to glucose
(AIRglu) by the acute glucose response after i.v.
glucose challenge. AIRglu adjusted for glucose
response was 90% lower in patients with T2D
compared with control participants. This difference
between groups was similar to the results without
adjustment for glucose levels.
The plasma insulin and glucose levels had
returned to baseline values in both groups after
the arginine challenge and before administration of

M. Sj
ostrand et al.

The arginine stimulation test in young patients with T2D

Table 1 Acute insulin (AIR) and C-peptide (Ac-pepR) responses after i.v. glucose and arginine administration and HOMA-b,
HOMA-IR, fasting C-peptide and proinsulin/insulin ratio in study groups
Control (n = 23)

Type 2 diabetes (n = 54)

P-value

AIRglu, pmol L

558  360

Ac-pepRglu, nmol L

1.40  0.79

0.23  0.50

AIRarg, pmol L

426  258

306  264

<0.01

Ac-pepRarg, nmol L

1.10  0.46

0.93  0.60

<0.01

179  86

HOMA-b
1

Proinsulin/insulin

<0.001
<0.001

163  114

4.4  3.2

HOMA-IR
C-peptide, nmol L

60  156

Ns

11.0  8.2

<0.001

1.10  0.40

1.10  0.56

Ns

1.06  0.65

1.17  0.85

Ns

Data are mean  SD. AIR and Ac-pepR values are the mean of the three maximum values minus the prestimulus value.

Healthy

(a)

T2D

Insulin (pmol L1)

600
480
360
240
120

50

100

50

Healthy

(b)

Glucose (mmol L1)

150

100

150

Minutes

Glucose iv
Arginine iv

Glucose iv
Arginine iv

T2D

15

10

5
0

50

100

150

50

100

150

Minutes
Glucose iv
Arginine iv

Glucose iv
Arginine iv

Fig. 2 Acute insulin (a) and glucose (b) responses after i.v. glucose and arginine in healthy participants and patients with
type 2 diabetes (T2D).
2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

43

M. Sj
ostrand et al.

The arginine stimulation test in young patients with T2D

glucose (Fig. 2a, b). In addition, the C-peptide

concentration had returned to basal levels between
the arginine and glucose challenges (data not
shown). HOMA-b, proinsulin/insulin ratio and
fasting plasma C-peptide concentration did not
differ between the two groups. HOMA-IR was
significantly higher in patients with T2D than in
control participants (Table 1).
Correlations between fasting plasma glucose, proinsulin/insulin ratio
and C-peptide levels and insulin and C-peptide responses to i.v.
glucose and arginine
AIRglu and Ac-pepRglu, but not AIRarg and Ac-pepRarg,
responses were negatively correlated with fasting
p-glucose (r = 0.52 and
0.48, P < 0.05 and
r = 0.10 and 0.15, ns, respectively) in the T2D
group. The proinsulin/insulin ratio showed no
significant correlation with AIRglu and Ac-pepRglu
(r = 0.24 and 0.22) or with AIRarg and Ac-pepRarg
(r = 0.26 and 0.32). Fasting C-peptide was not
significantly correlated with AIRglu and Ac-pepRglu
(r = 0.11 and 0.14), but was positively correlated with AIRarg and Ac-pepRarg responses (r = 0.78
and 0.72, P < 0.05) in the T2D group. C-peptide,
insulin and proinsulin levels sampled at the time of
diabetes diagnosis were significantly and positively
correlated with AIRarg (r = 0.67, P < 0.05; r = 0.59,
P < 0.05; and r = 0.52, P < 0.05, respectively), but
not with AIRglu (r = 0.12,
0.13 and
0.12,
respectively). There was no significant correlation

between proinsulin/insulin ratio at diagnosis and

either AIRarg (r = 0.03) or AIRglu (r = 0.25). The
concentration of C-peptide at diagnosis was significantly correlated with the fasting C-peptide level
on the investigational day (r = 0.67, P < 0.0001).
Insulin and C-peptide responses after i.v. glucose and arginine,
HOMA-b and fasting C-peptide in subjects with different duration of
T2D
The insulin and C-peptide responses after arginine
administration were gradually reduced in the
groups with longer duration of T2D, whereas the
glucose-induced insulin and C-peptide responses
were similar irrespective of disease duration
(Table 2). These findings of the impact of diabetes
duration on arginine-stimulated insulin secretion,
as presented in Table 2, are further supported by
linear correlation analysis (P < 0.05). AIRarg and AcpepRarg were significantly negatively correlated with
diabetes duration (r = 0.41 and 0.47, P < 0.05),
whereas AIRglu and Ac-pepRglu showed no significant correlation (r = 0.13 and 0.14). Pretest
plasma glucose concentration was 7.8  1.8,
7.6  1.7 and 7.3  1.8 mmol L 1 (ns) in patients
with a diabetes duration of <5 years, 5 to <8 years
and 810 years, respectively. HOMA-b did not
differ significantly between the three patient
subgroups (Table 2). Patients with a disease duration of <5 years had significantly higher pretest Cpeptide levels than those in the two other groups

Table 2 Acute insulin (AIR) and C-peptide (Ac-pepR) responses after i.v. glucose and arginine, and HOMA-b, HOMA-IR and
fasting C-peptide, in subgroups based on diabetes duration
Diabetes duration (years)
<5 (n = 11)

5 to <8 (n = 20)

810 (n = 23)

P-value

AIRglu, pmol L

51.6  228.0

62.4  156.0

27.6  50.4

Ns

Ac-pepRglu, nmol L

0.13  0.66

0.23  0.50

0.10  0.23

AIRarg, pmol L

498  312

342  306

186  120

<0.05

Ac-pepRarg, nmol L

1.20  0.63

0.93  0.60

0.53  0.23

<0.05

Ns
810 years vs. <5 years
810 years vs. 5 to
<8 and <5 years

162  122

HOMA-b
HOMA-IR
C-peptide, nmol L

224  305

200  198

Ns

10.5  8.2

11.1  8.8

10.2  7.9

Ns

1.50  0.56

1.10  0.60

0.86  0.36

<0.05
<5 years vs.
5 to <8 and 810 years

Data are mean  SD.

44

2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

M. Sj
ostrand et al.

with longer
(Table 2).

The arginine stimulation test in young patients with T2D

duration

of

diabetes

(P < 0.05)

Insulin and C-peptide response after i.v. glucose and arginine, HOMAb and fasting C-peptide in subjects with T2D treated with diet/
exercise alone, OHAs and/or insulin
AIRglu and Ac-pepRglu were higher amongst patients
treated with diet/exercise alone compared with
those treated with OHAs, whereas the AIRarg and
Ac-pepRarg responses were approximately 6070%
lower in patients treated with insulin compared
with the other treatment groups (Table 3). The
results were essentially the same in a subanalysis
in which patients receiving OHAs other than metformin were excluded (data not shown). Pretest
plasma glucose concentration was 6.2  1.2,
7.8  1.7, 7.8  1.9 and 7.8  1.7 mmol L 1 in
the groups treated with diet/exercise alone, OHAs
and OHAs in combination with insulin and insulin
alone, respectively (significantly lower in the diet/
exercise-treated group versus all other treatment
groups, P < 0.05). Subjects treated with insulin
alone had significantly lower C-peptide levels than
those treated with OHAs (P < 0.05) or diet/exercise
alone (P < 0.05) (Table 3).

Discussion
The results of the present study indicate that the
i.v. arginine test can differentiate between subjects
with different duration and severity (as reflected by
different antidiabetic treatments) of disease. In
addition, the present data confirm that the arginine-stimulated insulin secretion response is less
influenced by the pretest plasma glucose level than
the insulin secretory response after administration
of glucose. Moreover, diabetes duration was negatively correlated with AIRarg and Ac-pepRarg, but not
with AIRglu and Ac-pepRglu.
The insulin and C-peptide responses after glucose
administration were decreased by approximately
90% in the T2D group, whereas the responses to
arginine were decreased by approximately 30%
compared with controls. It should be noted, however, that without controlling for baseline plasma
glucose levels or adjusting for apparent differences
in insulin sensitivity (i.e. HOMA-IR) between the
two study groups, the presently estimated relative
impairment of b-cell secretion in patients with T2D
may have been underestimated. The difference in
insulin secretory responses to i.v. arginine and

Table 3 Acute insulin (AIR) and C-peptide (Ac-pepR) responses after i.v. glucose and arginine, and HOMA-b, HOMA-IR and
fasting C-peptide, in subgroups based on treatment
Diabetes treatment
OHA + insulin

Insulin alone

n = 10

OHA n = 22

n = 12

n = 10

P-value

159.0  252.0

25.8  108.0

13.8  90.0

12.0  22.8

<0.05

0.46  0.79

0.07  0.33

0.15  0.30

0.05  0.12

<0.05

402  372

354  288

300  162

108  39.6

<0.05

0.96  0.66

0.93  0.60

0.83  0.40

0.40  0.17

<0.05

391  402

177  138

<0.05

9.6  5.0

<0.05

Diet/exercise
AIRglu, pmol L

diet/exercise vs. tablets

Ac-pepRglu, nmol L

diet/exercise vs. tablets

AIRarg, pmol L

insulin vs. other treatments

Ac-pepRarg, nmol L

insulin vs. other treatments

HOMA-b

173  126

HOMA-IR

6.2  5.4

121  78

tablets + insulin vs. tablets

8.4  6.2

19.1  9.9

tablets + insulin vs. diet/

exercise and tablets
C-peptide, nmol L

1.20  0.73

1.26  0.46

1.00  0.53

0.60  0.17

<0.05
insulin vs. diet/exercise and
tablets

Data are mean  SD.

2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

45

M. Sj
ostrand et al.

glucose stimulation in patients with T2D, on the

other hand, cannot be attributed to variations in
glucose levels, as plasma glucose was comparable
before the two challenge tests. It is well known
that the glucose-dependent first-phase insulin
response is impaired in T2D and in states of
increased plasma glucose levels [10, 12, 13] due
to glucose toxicity. Indeed, in the present study,
fasting plasma glucose was negatively correlated
with the acute insulin and C-peptide response after
a glucose load (even though fasting glucose level
was required to be in the range 412 mmol L 1),
whereas no correlation was found between fasting
plasma glucose and the acute insulin and Cpeptide response to arginine. Thus, our data may
indicate that AIRarg and Ac-pepRarg are better tests
of the functional b-cell reserve than AIRglu and
Ac-pepRglu in patients with T2D with moderately
increased fasting plasma glucose levels. This is in
agreement with earlier findings [9], in patients who
have undergone pancreas transplantation, that
AIRarg and Ac-pepRarg are less dependent on baseline plasma glucose levels. In addition, AIRarg has
been shown to be a better predictor than AIRglu of
b-cell secretory capacity in subjects found to be
positive for islet-cell antibodies during progression
to manifest type 1 diabetes and in those with type 1
diabetes who received pancreatic islet-cell transplantation [14, 15]. Rodent experimental models
have also demonstrated that AIRglu is reduced to a
larger extent than AIRarg following partial pancreatectomy [16]. The glucose-stimulated insulin
secretion was higher in the diet/exercise-treated
patients with T2D than in the other antidiabetic
treatment groups. This may be partly explained by
the lower fasting glucose levels (and a lesser degree
of glucose toxicity) before the stimulation test
compared with the groups receiving pharmacological antidiabetic treatment (who had similar pretest
fasting glucose levels). On the other hand, the
arginine-stimulated insulin secretion decreased
more gradually in parallel with escalating therapies
and was most clearly reduced in patients treated
with insulin alone. This may indicate that the
arginine test is more sensitive to different degrees
of b-cell dysfunction at later stages of the disease
than the i.v glucose tolerance test (IVGTT).
In the present study, the often-used index of b-cell
function, HOMA-b, did not differ between individuals with T2D and matched healthy control participants, although HOMA-IR was significantly higher
in the T2D group. This may be explained by the fact
that fasting insulin concentration (included in the
46

2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

The arginine stimulation test in young patients with T2D

estimation of both HOMA-b and HOMA-IR) is

strongly influenced by both b-cell function/mass
and peripheral insulin resistance. This is supported
by the recent finding that b-cell area was predicted
by a b-cell function test (C-peptide/glucose ratio
after OGTT), but not by HOMA-b index in patients
with chronic pancreatitis or pancreatic tumours
undergoing surgery [17]. The use of HOMA-b as a
reliable measure of b-cell function has previously
been questioned [18] and, in particular, its use in
cohorts with TD2 treated with insulin and/or insulin secretagogues (as the relationship between fasting glucose and insulin is altered in the diseases) is
controversial [7]. Pharmacological glucose-lowering
treatments affecting insulin secretion, for example
sulphonylureas, will also skew the HOMA measures. In the current study, there was a large
variation in both HOMA-b and HOMA-IR, especially in the T2D group, possibly because approximately 40% of the subjects with T2D in this
study were treated with insulin, which could affect
the fasting insulin levels. Taken together, our
findings confirm that HOMA-b is not a reliable
measure of b-cell function in most patients with
manifest T2D. Also, the lack of difference in proinsulin/insulin ratio between patients with T2D and
control participants was probably due to the fact
that a high proportion of the patients were treated
with insulin.
Plasma C-peptide levels both at diagnosis of diabetes and on the day of the investigation were
correlated
with
arginine-stimulated
insulin
release. In addition, fasting C-peptide declined
with longer duration of T2D and so may have the
potential to serve as a surrogate marker of b-cell
function. However, fasting C-peptide levels did not
differ between the T2D and healthy control groups.
Therefore, overall, the role of C-peptide as a marker
for functional b-cell mass remains unclear. It has
previously been reported that b-cell function (measured as C-peptide concentration) decreases over
time in patients with T2D, but the rate of decline
varies between individuals [2]. Similar to HOMA
indices and insulin levels, C-peptide will be related
to insulin resistance, and again, on its own not
likely to reflect b-cell function, as the degree of
insulin resistance needs to be taken into account
as well [19].
Several limitations of this study should be considered. First, because of the relatively small number
of patients in the T2D subgroups, type II errors
cannot be excluded, and thus, the power to detect

M. Sj
ostrand et al.

true differences between subgroups is limited.

Notably, the T2D patients were confirmed by the
absence of islet-cell antibodies. A second limitation
is the cross-sectional design; therefore, longitudinal studies to follow the change in functional b-cell
mass (e.g. measured as AIRarg and Ac-pepRarg) over
time are needed. Thirdly, the T2D group in the
current study was relatively young with an early
onset of diabetes; thus, our finding of b-cell
dysfunction may not be generalizable and should
be confirmed in a patient population with a
broader, more typical age range.
Another limitation of this study is the short washout period of ongoing antidiabetic treatments, so
that pharmacological effects could remain and
affect the measures of b-cell function. However,
agents that directly stimulate insulin secretion,
that is sulfonylureas, were short acting, and exposures should be below therapeutic levels at the
time of the investigation. In this study, differences
between the groups remained essentially the same
after excluding patients treated with OADs other
than metformin. In addition, 40% of the patients
were treated with insulin, and residual insulin
from long-acting preparations taken the night
before, which could still remain in the circulation
at the time of testing, may have influenced the
insulin secretory responses. However, when
estimating the dynamic b-cell function with the
i.v. arginine and glucose test, the changes in
insulin and C-peptide levels are used, which may
be assumed to be less affected than the fasting
levels.
Hyperglycaemic and euglycaemic clamps are the
gold standard method to measure b-cell function
and insulin sensitivity, and fasting glucose levels
should be normalized by an overnight infusion of
insulin. However, in the clinical setting, less timeconsuming and labour-intensive methods are warranted. In theory, the insulin secretory response
after i.v. glucose administration may also have
been affected by the preceding arginine stimulus,
as arginine may modify the responsiveness of the
b-cells to subsequent stimulation (the memory of
the b-cell) [20]. As reviewed previously [21], experimental studies using the perfused isolated rat
pancreas have shown that arginine time-dependently reduces the insulin response to subsequent
stimulation. Following combined challenge with
arginine and glucose, however, there is no inhibition of the second response. Accordingly, studies in
vivo in healthy participants and patients with T2D

The arginine stimulation test in young patients with T2D

have shown that the priming effect of arginine is

small at normal plasma glucose concentrations
and absent at moderate levels of hyperglycaemia
[22, 23].
In conclusion, b-cell secretory capacity measured
with the arginine stimulation test (AIRarg and AcpepRarg) was less attenuated in patients with T2D,
compared with healthy control participants, and
was gradually reduced with longer disease duration and intensified antidiabetic treatment. We
confirmed that the first phase of insulin secretion
after a glucose load was markedly attenuated in
young patients with T2D compared with healthy
control participants, but did not seem to differ
between patients with different stages of disease. In
addition, in contrast to arginine-stimulated insulin
secretion, insulin secretion after a glucose load
was highly dependent on and inversely correlated
with the baseline plasma glucose level. Furthermore, HOMA-b was an unreliable measure of b-cell
function in this study population, and plasma
C-peptide was not able to differentiate between
patients with T2D and healthy control participants.
Although further validation is required, the arginine test may prove to be useful in epidemiological
and clinical studies for identification of T2D
patients with different degrees of b-cell dysfunction. It might also be valuable for selection and
follow-up of patients in trials of interventions to
preserve or restore b-cell function and mass.
Author contributions
M.S. wrote the manuscript, designed the study and
participated in the interpretation of data. J.B and
J.W.E designed the study, participated in the
interpretation of data, contributed to the discussion and edited/reviewed the manuscript. K.C did
the statistical analysis of the data. S.L participated
in acquisition of data. M.K.S, M. L-O, S.G, H.J.A
and L.N contributed to the design of the study,
participated in acquisition and interpretation of
data and reviewed the manuscript.
Conflict of interest statement
JB has received consulting and/or lecture fees
from AstraZeneca. MS, JWE and KC are full-time
employees of AstraZeneca. MKS, ML-O SG, SL,
HJA and LN have no conflict of interests to declare.
The study was funded by AstraZeneca.
2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

47

M. Sj
ostrand et al.

Acknowledgement
Professor Bj
orn C Carlsson at AstraZeneca is the
guarantor of the study.

References
1 U.K. Prospective Diabetes Study Group. U.K. prospective
diabetes study 16. Overview of 6 years therapy of type II
diabetes: a progressive disease. U.K. Prospective Diabetes
Study Group. Diabetes 1995; 44: 124958.
2 Zangeneh F, Arora PS, Dyck PJ et al. Effects of duration of
type 2 diabetes mellitus on insulin secretion. Endocr Pract
2006; 12: 38893.
3 DAlessio DA, Denney AM, Hermiller LM et al. Treatment with
the dipeptidyl peptidase-4 inhibitor vildagliptin improves
fasting islet-cell function in subjects with type 2 diabetes. J
Clin Endocrinol Metab 2009; 94: 818.
4 Kahn SE, Lachin JM, Zinman B et al. Effects of rosiglitazone,
glyburide, and metformin on b-cell function and Insulin
sensitivity in ADOPT. Diabetes 2011; 60: 155260.
5 Bunck MC, Diamant M, Corn
er A et al. One-year treatment
with exenatide improves b-cell function, compared with
insulin glargine, in metformin-treated type 2 diabetic
patients. Diabetes Care 2009; 32: 7628.
6 Ahr
en B. GLP-1 for type 2 diabetes. Exp Cell Res 2011; 317:
123945.
7 Wallace TM, Levy JC, Matthews DR. Use and Abuse of HOMA
Modeling. Diabetes Care 2004; 27: 148795.
8 Ward WK, Bolgiano DC, McKnight B, Halter JB, Porte D Jr.
Diminished B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus. J Clin Invest 1984; 74:
131828.
9 Robertson RP. AIRarg and AIRgluc as predictors of insulin
secretory reserve. Transplant Proc 2004; 36: 10401.
10 Robertson RP. Consequences on b-cell function and reserve
after long-term pancreas transplantation. Diabetes 2004; 53:
63344.
11 Borg H, Arnqvist HJ, Bjork E et al. Evaluation of the
new ADA and WHO criteria for classification of diabetes
mellitus in young adult people (15-34 yrs) in the Diabetes
Incidence Study in Sweden (DISS). Diabetologia 2003; 46:
17381.
12 Brunzell JD, Robertson RP, Lerner RL et al. Relationships
between fasting plasma glucose levels and insulin secretion
during intravenous glucose tolerance tests. J Clin Endocrinol
Metab 1976; 42: 2229.
13 Ferrannini E. The stunned b cell: a brief history. Cell Metab
2010; 11: 34952.
14 Chaillous L, Rohmer V, Maugendre D et al. Differential
beta-cell response to glucose, glucagon, and arginine during
progression to type I (insulin-dependent) diabetes mellitus.
Metabolism 1996; 45: 30614.

48

2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2014, 275; 3948

The arginine stimulation test in young patients with T2D

15 Rickels MR, Naji A, Teff KL. Acute insulin responses to

glucose and arginine as predictors of beta-cell secretory
capacity in human islet transplantation. Transplantation
2007; 84: 135760.
16 Bonner-Weir S, Trent DF, Weir GC. Partial pancreatectomy in
the rat and subsequent defect in glucose-induced insulin
release. J Clin Invest 1983; 71: 1544.
17 Meier JJ, Menge BA, Breuer TGK et al. Functional assessment of pancreatic b-cell area in humans. Diabetes 2009; 58:
1595603.
18 Chang AM, Smith MJ, Bloem CJ, Galecki AT, Halter JB,
Supiano MA. Limitation of the homeostasis model assessment to predict insulin resistance and b-cell dysfunction in
older people. J Clin Endocrinol Metab 2006; 91: 62934.
19 Ahren B, Pacini G. Importance of quantifying insulin secretion in relation to insulin sensitivity to accurately assess beta
cell function in clinical studies. Eur J Endocrinol 2004; 150:
97104.
20 Grill V, Adamson U, Rundfeldt M, Andersson S, Cerasi E.
Glucose Memory of Pancreatic B and A2 Cells: evidence for
common time-dependent actions of glucose on insulin and
glucagon secretion in the perfused rat pancreas. J Clin Invest
1979; 64: 7007007.
21 Nesher R, Eylon L, Segal N, Cerasi E. b-cell memory to insulin
secretagogues: characterization of the time-dependent inhibitory control system in the isolated rat pancreas. Endocrinology 1989; 124: 1428.
22 Larsson H, Ahren B. Glucose-dependent arginine stimulation
test for characterization of islet function: studies on reproducibility and priming effect of arginine. Diabetologia 1998;
41: 7727.
23 Nesher R, Tuch B, Hage C, Levy J, Cerasi E. Time-dependent
inhibition of insulin release: suppression of the arginine effect
by hyperglycaemia. Diabetologia 1984; 26: 1425.
Correspondence: Mikaela Sj
ostrand, AstraZeneca R&D, Clinical
Development CVGI, Pepparedsleden 1, 431 83 M
olndal, Sweden.
(fax: +46 31 7763782; e-mail: mikaela.sjostrand@astrazeneca.
com).

Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Table S1. Patients characteristics in type 2 diabetes group (n = 54) divided on subgroups, based on
diabetes duration.
Table S2. Patient characteristics in type 2 diabetes
group (n = 54) divided on subgroups, based on
treatment.