Altered Glucose Homeostasis Is Associated with Increased Serum Apelin Levels in Type 2 Diabetes Mellitus
et al. (2012) Altered Glucose Homeostasis Is Associated with Increased Serum Apelin Levels
in Type 2 Diabetes Mellitus. PLoS ONE 7(12): e51236. doi:10.1371/journal.pone.0051236
Altered Glucose Homeostasis Is Associated with Increased Serum Apelin Levels in Type 2 Diabetes Mellitus
Maria Gisella Cavallo 0
Federica Sentinelli 0
Ilaria Barchetta 0
Carmine Costantino 0
Michela Incani 0
Laura Perra 0
Danila Capoccia 0
Stefano Romeo 0
Efisio Cossu 0
Frida Leonetti 0
Luciano Agati 0
Marco G. Baroni 0
Nigel Irwin, University of Ulster, United Kingdom
0 1 Endocrinology and Diabetes, Department of Medical Sciences, University of Cagliari , Cagliari , Italy , 2 Department of Internal Medicine and Medical Specialties, Sapienza University of Rome , Rome , Italy , 3 Department of Clinical Sciences, Sapienza University of Rome , Rome , Italy , 4 Department of Molecular and Clinical Medicine, Sahlgrenska Center for Cardiovascolar and Metabolic Research, University of Gothenburg , Gothenburg , Sweden , 5 Department of Cardiovascular Sciences, Sapienza University of Rome , Rome , Italy
Background: Apelin is an adipokine that plays a role in the regulation of glucose homeostasis and in obesity. The relationship between apelin serum concentration and dysmetabolic conditions such as type 2 diabetes (T2D) is still controversial. Aims of our study are: 1) determine the circulating levels of apelin in a large cohort of Italian subjects with T2D, T1D and in non-diabetic controls; 2) identify putative metabolic determinants of modified apelin concentrations, in order to search possible mechanism of apelin control; 3) investigate changes in apelin levels in response to sharp modifications of glucose/insulin metabolism in T2D obese subjects before and 3 days after bariatric surgery. Methods: We recruited 369 subjects, 119 with T2D, 113 with T1D and 137 non-diabetic controls. All subjects underwent a complete clinical examination, including anthropometric and laboratory measurements. Serum apelin levels were determined by EIA (immunoenzyme assay). Results: Patients with T2D had significantly higher serum apelin levels compared to controls (1.2361.1 ng/mL vs 0.9160.7 ng/mL, P,0.001) and to T1D subjects (0.7360.39 ng/mL, P,0.001). Controls and T1D subjects did not differ significantly in apelin levels. Apelin concentrations were directly associated with fasting blood glucose (FBG), body mass index (BMI), basal Disposition Index (DI-0), age, and diagnosis of T2D at bivariate correlation analysis. Multiple regression analysis confirmed that diagnosis of T2D, basal DI-0 and FBG were all determinants of serum apelin levels independently from age and BMI. Bariatric surgery performed in a subgroup of obese diabetic subjects (n = 12) resulted in a significant reduction of apelin concentrations compared to baseline levels (P = 0.01). Conclusions: Our study demonstrates that T2D, but not T1D, is associated with increased serum apelin levels compared to non-diabetic subjects. This association is dependent on impaired glucose homeostasis, and disappears after bariatric surgery, providing further evidence regarding the relationship between apelin and the regulation of glucose metabolism.
Funding: This work was funded by research grants from the University of Cagliari (ex-60% 200911 http://www.unica.it/), from the Ministry of Education,
University and Research (PRIN 2008, n. 2008J2FS4T, http://prin.cineca.it/) and from the Foundation Banco di Sardegna (Research Projects 2011, http://www.
fondazionebancodisardegna.it/), all awarded to MGB. MGC was funded by a research grant from Sapienza University of Rome (http://www.uniroma1.it/) The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Insulin resistance is a major characteristic of type 2 diabetes
mellitus (T2D) and is often linked to obesity . In combination,
these events increase the risk of cardiovascular diseases and
obesity-associated morbidity. Excess in adipose tissue plays a
central role in the induction of insulin-resistance.
In the last decade, many different studies demonstrated the
existence of a number of adipocyte-derived secretory factors
(adipokines) which take part in the control of body energy
homeostasis and glucose metabolism . Among them, a new
peptide has been recently identified, named apelin (also known as
APJ receptor ligand) . Boucher et al. demonstrated that apelin is
produced and secreted by both human and mouse white adipose
tissue, acting therefore as an adipokine . Apelin mRNA is
detectable in non-differentiated preadipocytes and its production
increases 4-fold upon differentiation of the fat cells, as previously
found for adiponectin and leptin . In humans, apelin gene is
widely expressed in adipose tissue, heart, stomach, placenta and
breast, as well as in different brain areas, suggesting an important
role of this molecule also in the central regulation of metabolic
Native preproapelin exists as a dimer of 77 amino acids that is
cleaved into active forms of C-terminal fragments, including
apelin-36, apelin-17, apelin-13, and the post-translationally
modified (Pyr1)apelin-13 and apelin-12 . All of these predicted
isoforms have been shown to be present in vivo. Apelin-12 is the
smallest C-terminal fragment to bind and activate the apelin
receptor , and any apelin fragment containing this 12 amino
acid core maintains all bioactivity.
Findings from several studies suggest that apelin treatment
during insulin resistance triggers a number of coordinated
beneficial effects, including reduction of hyperinsulinemia and
adiposity, and stimulation of glucose uptake and fuel consumption
. Insulin resistance in muscle is characterized by impaired
glucose uptake, reduced glycogen synthesis, insufficient fat
oxidation, fat accumulation and cellular stress. In skeletal muscle
apelin has been shown to improve the overall insulin-sensitivity,
both in vitro and in animal models . In adipose tissue apelin
infusion in apelin2/2 mice decreased adiposity and FFAs and also
glycerol levels, suggesting a role for apelin in the regulation of
lipolysis . However, in human adipose tissue explants or in
human isolated adipocytes, apelin had no effect on basal or
isoproterenol-stimulated lipolysis . Finally, in pancreas apelin
was also shown to inhibit both glucose-induced and glucagon- like
peptide 1 (GLP-1) stimulated insulin secretion in INS-1 cells ,
indicating that apelin acts as a regulator of insulin-secretion.
In humans, evidences on apelin regulation in presence of
impaired glucose metabolism are still controversial. Some studies
found increased apelin levels in very small populations of obese
patients with impaired glucose tolerance or T2D [13;14]. On the
opposite, other authors surprisingly reported low apelin levels in
obese subjects with newly diagnosed T2D compared to
nondiabetic individuals [15;16]. In a small sample of children with
type 1 diabetes apelin levels were reported to be increased
compared to healthy controls . In the only large study in
patients with gestational diabetes no difference was found in apelin
levels between patients and control women . Thus more
consistent data are warranted.
Bariatric surgery could be viewed as a model of diabetes
remission. To our knowledge, only one study compared serum
apelin concentrations before and after bariatric surgery, showing a
significant reduction only in patients affected by impaired glucose
regulation or T2D before surgery .
Thus, aims of our study are: 1) to determine serum apelin levels
in a large cohort of Italian subjects with T2D, T1D and in
nondiabetic controls; 2) to identify putative metabolic determinants of
modified apelin concentrations, in order to explore possible
mechanism of apelin control; 3) to explore changes in apelin levels
in response to sharp modifications of glucose/insulin metabolism
in T2D obese subjects before and 3 days after bariatric surgery.
This study was reviewed and approved by the Ethics Committee
of Policlinico Umberto I, Sapienza University of Rome and
conducted in conformance with the Helsinki Declaration.
Written consent was obtained from all patients before the study.
For these purposes, we recruited 369 subjects, 119 affected by
T2D (mean age6SD: 61610 years), 113 with T1D (mean
age6SD: 25614 years), and 137 non-diabetic controls (mean
age6SD: 49610 years), among subjects attending the Internal
Medicine out-patient clinics of Sapienza University of Rome. All
subjects had a complete work-up including clinical examination,
anthropometric measurements and laboratory tests.
Study population underwent fasting blood sampling to assess
FBG, glycosylated hemoglobin (HbA1c), total cholesterol,
HDLcholesterol, triglycerides, aspartate aminotransferase (AST),
alanine aminotransferase (ALT), nitrogen and creatinine by standard
laboratory methods. Insulin was measured by radio-immuno-assay
(ADVIA Insulin Ready Pack 100, Bayer Diagnostics, Milan, Italy),
with intra- and inter-assay coefficients of variation ,5%.
Lowdensity lipoprotein (LDL) cholesterol value was obtained using
Friedwald formula. The homeostasis model assessment of insulin
resistance (HOMA-IR) was calculated as previously described
. Metabolic Syndrome (MS) was defined according to
modified NCEP ATP-III criteria  and diabetes mellitus
according to ADA 2009 criteria .
Basal disposition index (DI-0), which gives an adjusted measure
of insulin sensitivity according to the HOMA of insulin resistance
(HOMA-IR) is calculated by using the formula DI-0 = HOMA-B*
(1/HOMA-IR) . The DI is a measure of the ability of the
betacells to compensate for insulin resistance. It can be considered a
measure of the functionality of the pancreas and can predict the
normal beta-cell response adequate for any degree of insulin
resistance . In diabetes, beta-cells are unable to respond
adequately to insulin-resistance, thus determining the appearance
of impaired glucose regulation and altering the Disposition Index.
Apelin-12 levels were measured by a non-selective
enzymelinked immunosorbent assay (ELISA) kits (EK-057-23; Phoenix
Pharmaceutical, Inc. Belmont, CA, USA) on sera frozen
immediately after separation and stored at 225uC. The
Apelin12 EIA Kit presents 100% cross-reactivity with human Apelin-12,
Apelin-13 and Apelin-36. The sensitivity of the technique was
0.06 ng/mL and the intra-assay and inter-assay CV% reported by
the manufacturer were 510% and ,15%, respectively.
SPSS version 17 statistical package was used to perform the
analyses. These included Students t test for normally-distributed
variables, Mann-Whitney non-parametric independent sample test
and x2 test for categorical variables, as appropriate. Correlations
were estimate by Spearmans rho non parametric test. The
Wilcoxons rank test for paired samples was used to compared
clinical and biochemical parameters of T2D patients before and
after bariatric surgery.
A multiple liner regression analysis was performed to investigate
independent association between serum apelin levels (dependent
variable) and selected variables that had p-values ,0.05 in
univariate analysis (sex and age were also included). P-values
,0.05 were considered statistically significant with a confidence
interval of 95%.
We performed a sample size calculation based on the standard
deviation (SD) of apelin levels in controls (SD = 0.70 ng/mL). If
the true difference in the experimental and control means was
0.30, we needed to study 86 cases affected by diabetes vs 86
subjects without diabetes to be able to reject the null hypothesis
that the population means of the patients and the controls were
equal with probability (power) 0.80. The Type 1 error probability
associated with this test is 0.05.
Fasting blood glucose (mg/dL)
Fasting blood insulin (mU/mL)
Diabetes duration (years)
Data are expressed as mean 6 standard deviation. Mann-Whitney non-parametric independent sample test was applied. P values ,0.05 are considered significant.
Abbreviations : BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; DI-0 basal disposition index; HbA1c, glycated hemoglobin A1c;
HOMAIR, homeostatic model assessment of insulin resistance; HDL, high density lipoprotein; LDL, low density lipoprotein; AST, aspartate aminotransferase; ALT, alanine
Clinical and metabolic characteristics of study population are
summarized in Table 1. Briefly, patients affected by T2D had
significantly higher age, FBG, fasting insulin, BMI, HbA1c,
HOMA-IR, total-cholesterol, LDL-cholesterol, transaminases,
systolic and diastolic blood pressure and lower HDL-cholesterol
compared to non-diabetic control, as expected. T1D subjects had
significantly higher glucose, HbA1c, and lower age, BMI,
triglycerides compared to non-diabetic controls.
Serum apelin levels were significantly higher in T2D patients
compared to controls (1.2361.1 ng/mL vs 0.9160.7 ng/mL,
P,0.001) and to T1D subjects (0.7360.39 ng/mL P,0.001). No
difference was observed in apelin levels between controls and T1D
subjects (P = NS) (Table 1).
Correlation of Apelin with Metabolic and Clinical
Spearmans correlation analyses in our population of T2D and
non-diabetic controls (without T1D) demonstrated that serum
apelin levels were directly associated with FBG, BMI, age and
diagnosis of T2D, and negatively associated with basal Disposition
Index (DI-0), whereas no association was found with other clinical
and metabolic parameters (Table 2). In T1D apelin levels
negatively correlated with HbA1c and positively with duration of
the disease (data not shown).
Multivariate Analysis of Apelin Determinants
Multiple regression analysis with all the significant variables
from the Spearmans correlation analyses confirmed that diagnosis
of T2D, basal Disposition Index and fasting plasma glucose were
all determinants of serum apelin levels independently from age,
and BMI (P = 0.04) (Table 3). Adjustment for sex did not change
In order to further investigate the relative influence of BMI on
serum apelin levels, we stratified our whole population (T2D and
controls) in three groups of normal-weight (BMI = 2024.9 Kg/
m2), overweight (BMI: 2529.9 Kg/m2) and obese (BMI.30 Kg/
m2) subjects and found significantly increased apelin levels in
presence of higher BMI (0.8260.58 ng/mL in normal-weight,
1.0160.87 ng/mL in overweight, 1.361.2 ng/mL in obese
subjects, trend test P = 0.03). Then, the study population was
grouped according to the presence/absence of T2D and we
observed that, among the obese subgroup (BMI.30 Kg/m2),
patients with T2D had significantly higher apelin levels than
nondiabetic subjects (1.6261.43 ng/mL vs 0.9760.78 ng/mL,
P = 0.015), thus confirming the strong independent association of
diabetes with apelin levels.
Antidiabetic Treatment and Apelin Levels
We also analysed the possible influence of antidiabetic
treatment on apelin levels. Seventy-three percent (n = 87) of our
T2D patients were treated with oral antidiabetic drugs, with 77%
of them receiving metformin alone or in combination, and only a
few (n = 14) were insulin-treated. Apelin levels were not
signifiFasting blood glucose
Fasting blood insulin
Spearmans correlation coefficient. P values ,0.05 are considered significant.
Abbreviations: BMI, body mass index; SBP, systolic blood pressure; DBP,
diastolic blood pressure; DI-0 basal disposition index; HbA1c, glycated
hemoglobin A1c; HOMA-IR, homeostatic model assessment of insulin
resistance; HDL, high density lipoprotein; LDL, low density lipoprotein; AST,
aspartate aminotranferase; ALT, alanine aminotransferase; MS, metabolic
Apelin Levels and Bariatric Surgery
Finally, we explored serum apelin concentration changes after
bariatric surgery in a subgroup of obese patients affected by T2D
(n = 12), who underwent laparoscopic sleeve gastrectomy for
weight reduction. Three days after surgery, when metabolic
changes are already evident but before any significant reduction in
weight and BMI, apelin levels were significantly decreased
compared to baseline (2.0362.5 vs 0.8660.44 ng/mL,
P = 0.013, respectively). T2D obese subjects showed also a
significant reduction in fasting blood glucose (from 145.6 to
110.2 mg/dL, P,0.008), and an increase in DI-0 (22.3620.3 vs.
46.5632.02, P,0.008). Nine (75%) of these patients were without
diabetes already after 3 days (Table 4 summarizes clinical
characteristics of this population before and after the intervention).
No significant difference was observed in apelin levels before and
after bariatric surgery (1.8360.54 vs 1.6360.31 ng/mL,
P = 0.279, respectively) in those 3 subjects that remained with
type 2 diabetes, with fasting blood glucose 165628 mg/dL.
To analyze if the reduction of apelin persisted after a longer
period of observation, we evaluated apelin levels in sera from a
subgroup of our diabetic obese subjects (n = 10), 6 months after
bariatric surgery. Apelin levels remained low, showing only a
modest non-significant (P = 0.55) increase compared to 3 days
levels, (1.2060.41 vs 0.8660.44 ng/mL, respectively). This
increase followed the modest increase observed in fasting glucose
levels (from 110 to 119 mg/dL, P = NS). None of these subjects
returned diabetic, but some (n = 3) showed fasting levels close to
the diabetic threshold. Moreover, apelin levels after 6 months did
not show any relationship with the important reduction in BMI
observed in these patients (from 40.8 to 31.5 Kg/m2 P,0.001).
The results of this study demonstrate, in a large cohort of Italian
subjects, that patients with type 2 diabetes have significantly
increased serum apelin levels compared to non-diabetic
individuals. The relationship between T2D and apelin is dependent on
the presence of alterations in glucose homeostasis but independent
from BMI and other metabolic abnormalities.
We also show for the first time that a key determinant of serum
apelin levels is the basal disposition index, a surrogate measure of
the two major defects in type 2 diabetes, defective insulin secretion
and impaired insulin-sensitivity. Further support on this
relationship is given by the low levels of apelin observed in type 1 diabetic
subjects, where insulin secretion is absent and insulin-resistance is
usually not present. Also Dray observed increased apelin
concentrations in insulin-resistant type 2 diabetic patients, and
this increase correlated positively with insulin levels. The
compensatory role of apelin in glucose homeostasis was also
confirmed by the phenotype of apelin null mice that are
hyperinsulinemic and insulin resistant. It was shown that apelin
treatment in these insulin-resistant obese-mice improves insulin
sensitivity . All together, these data may indicate that the
increased apelin levels that we observe in T2D patients could
represent a compensatory mechanism to reduce both insulin
resistance and impaired insulin-secretion. Alternatively, the apelin
increase may be due to a so called apelin-resistance, driven by yet
unknown mechanism determining this effect.
In our study we do not observe significant correlation between
apelin-12 concentration and HOMA-IR. The absence of
association with HOMA-IR may be explained by the observation that
the strongest association is found with type 2 diabetes, were both
insulin resistance and impaired insulin secretion are present and
necessary. Thus, it is possible that HOMA-IR alone cannot
determine the increase of apelin in diabetic subjects. The
Pre-surgery (n = 12)
3 days Post-surgery(n = 12)
Fasting blood glucose (mg/dL)
Fasting blood insulin (mU/mL)
Results are shown as Mean6SD. Non-parametric Wilcoxon Signed Rank test applied. P values ,0.05 are considered significant.
disposition index, representing both defective insulin secretion and
sensitivity, describes more effectively the presence of both defects.
In a previous study Boucher et al. showed a significant increase
in apelin plasma concentrations in different mouse models of
obesity associated with hyperinsulinemia, but not in the
nonhyperinsulinemic obese mouse. Furthermore, in non-diabetic
obese patients both plasma apelin and insulin levels were
significantly higher than lean controls, suggesting that insulin
could influence blood concentrations of apelin . In our study we
were unable to find a significant correlation between apelin and
insulin levels, but we should point out that only 15% of our T2D
patients were untreated, with most of these subjects taking
metformin and/or sulphonylureas or insulin.
Consistent with previous reports, we observed a significant
increase in apelin levels with increasing BMI. We also observed
that obese patients with type 2 DM had significantly higher apelin
levels than non-diabetic obese subject (1.6261.43 ng/mL vs
0.9760.78 ng/mL, P = 0.015) confirming that increased apelin
levels are directly associated with the presence of diabetes rather
than obesity itself.
With regards to the association between apelin and diabetes, in
a very small study, Dray at al. also found increased apelin plasma
levels in diabetic patients (n = 12) compared to controls (n = 11), in
line with our observations in a larger cohort . In contrast,
when apelin was measured in untreated T2D at the time of
diagnosis, its levels were found to be reduced compared to healthy
controls [15,16]. It may be postulated that a longer duration of
diabetes might worsen insulin resistance and secretion, further
influencing apelin levels. Newly diagnosed diabetic patients are in
the early stage of their natural history, when the metabolic defects
are not fully expressed, as shown by the possible reversal of T2D
with lifestyle measures. Therefore we suggested that differences in
diabetes duration may underlie discrepancies between studies.
In patients with type 1 diabetes we observed levels of apelin
comparable to non-diabetic controls. In the only other study in
children with T1D  the authors observed significantly
increased apelin levels compared to age-matched controls. In an
attempt to explain this discrepancies, we noticed that in this study
the reported standard deviations for apelin levels were 24 times
higher than those found in other studies, thus with a measurement
not comparable, and that the sample was much smaller and
younger than in our study.
In our study, a subgroup (n = 12) of our obese type 2 diabetes
subjects underwent laparoscopic sleeve gastrectomy for weight
reduction. Three days post surgery, a significant reduction in
apelin, fasting blood glucose and basal disposition index was
observed, together with a concomitant very small, non-significant,
BMI reduction. Moreover, insulin levels after bariatric surgery
showed a strong decrease, although not significant. The choice of
selecting samples 3 days after laparoscopic sleeve gastrectomy was
taken in order to observe possible changes in apelin levels before
any significant weight loss would occur. Therefore, given that
there were no significant differences in BMI at 3 days post-surgery,
we could speculate that apelin reduction may be due to the
changes observed in insulin sensitivity and insulin secretion
(measured by the Disposition Index) after bariatric surgery rather
than to a reduction of the adipose tissue mass. A further possible
explanation may be ascribed to the changes in blood glucose, with
remission of diabetes in 75% of the cases after 3 days post-surgery.
Although the study was not designed to evaluate long-term effects,
we were able to analyse 10 samples from the bariatric surgery
group that were collected after 6 months. Lower levels of apelin
compared to baseline persisted after six months from surgery and
were not related to the highly significant reduction in BMI
A recent association between apelin and TNF-alpha has been
reported  in subjects with the metabolic syndrome. Also,
Daviaud et al.  reported a significant association between
TNF-alpha and apelin in adipose tissue of a mouse model of
obesity. Since TNF-alpha is a marker of low-grade inflammation,
which is present in the insulin-resistant state, it could be also seen
as a marker of insulin-resistance, as it is for apelin. Although
inflammation may still be present, we hypothesize that the
dramatic metabolic changes observed in our subjects are probably
the main determinants of apelin changes.
Soriguer et al. found increased apelin levels in morbidly obese
patients with type 2 diabetes and a correlation between
prebariatric surgery apelin plasma levels and BMI in diabetic patients
. Moreover, after surgery, they could observe a significant
decrease of apelin levels only in the morbidly obese subjects with
impaired fasting glucose or diabetes, a decrease that followed the
changes in blood glucose and insulin sensitivity. These results are
in line with our observation that BMI is not an independent
determinant of high apelin levels, whereas diabetes is directly
associated to increased apelin, regardless to other metabolic
The surgical technique that was used in our patients was the
Laparoscopic Sleeve Gastrectomy, that deeply alters the complex
balance of gastrointestinal hormones, such as ghrelin, leptin,
GLP1, peptide YY (PYY) and pancreatic polypeptide (PP) [27,28].
Changes in these peptides appear to be critical for improving the
response to insulin. However, we cannot find experimental data
linking directly these peptides to apelin. It is possible that the
whole system, greatly modified by the surgical intervention, acts to
improve insulin-sensitivity and, as a consequence, to reduce apelin
Based on published data and on our own observations, the
pharmacological utility of apelin appears potentially useful. So far,
the only evidences available are in animal models, where apelin
administration, both long and short-term, improves insulin
sensitivity and glucose regulation in different animal model
[24,9]. Furthermore, given the pleiotropic effects on numerous
organs and tissues that are ascribed to apelin, the consequence of
chronic or acute apelin administration in humans on
cardiovascular functions, on central nervous system, and on glucose or lipid
metabolism needs to be evaluated. The development of ligands
agonists or antagonists versus apelin receptor are certainly
In conclusion, we investigated serum apelin levels in a large
cohort of Italian subjects with T2D compared to non-diabetic
controls and to patients with T1D. We demonstrated that T2D is a
determinant of increased circulating apelin levels independently
from the concomitant presence of obesity and other metabolic
alterations. Apelin is increased in relation to the worsening of
insulin-resistance/insulin-secretion, as in the diabetic status, and
this increase has been suggested as a possible compensatory
mechanism. Also, apelin levels, 3 days after bariatric surgery,
significantly decreased together with an improved metabolic
profile and independently from weight loss. This reduction
persisted after six month, and was still unrelated to weight loss.
Further studies are warranted to clarify the pathophysiological role
of apelin in whole body energy homeostasis and in metabolic
Conceived and designed the experiments: MGC FL LA MGB. Performed
the experiments: FS IB CC MI LP. Analyzed the data: FS IB MGC MGB
SR. Contributed reagents/materials/analysis tools: LP CC EC DC SR.
Wrote the paper: FS IB MGC MGB.
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