INTRODUCTION
Type 2 diabetes mellitus (T2DM) is a disease characterized by a combination of
insulin resistance and relative insulin insufficiency [
1,
2]. Obesity is one of the
most important factors associated with insulin resistance, which is the initial step
in the development of T2DM. The recent rapidly increasing prevalence of T2DM and
obesity has been identified as a major worldwide health crisis [
3]. Therefore, improving the understanding of
the various factors associated with this health issue is a very important focus of
research.
Glypican-4 (GPC4) is a member of the glycosylphosphatidylinositol (GPI)-anchored
heparin sulfate proteoglycans [
4]. GPC4 is
expressed in visceral and subcutaneous adipose tissues and was identified recently
as a novel adipokine [
5]. GPC4 was
demonstrated to regulate insulin signaling through interaction with the insulin
receptor and by inducing differentiation of adipocytes, indicating a potentially
important role in the regulation of body fat [
6,
7]. GPC4 also acts as an
essential modulator of key regulatory proteins, including Wnt, bone morphogenetic
proteins, fibroblast growth factor, and sonic hedgehog [
4]. GPC4 was also shown recently to play an important role in
fat distribution, an effect that was modified by rosiglitazone through differential
regulation of GPC4 mRNA in subcutaneous and visceral fat tissues [
7]. In addition, studies of GPC4 expression in
human white adipose tissue showed a positive correlation with body fat content and
insulin resistance [
6], while the circulating
plasma GPC4 level was increased in women but not in men with nonalcoholic fatty
liver disease (NAFLD) [
8]. In another study,
GPC4 was higher in patients with prediabetes than that in normal subjects [
9]. These studies showed that GPC4 level was
significantly elevated in states of metabolic dysfunction, including obesity,
insulin resistance, and NAFLD.
There has been no definitive study of GPC4 level and its correlation with other
clinical factors in typical T2DM patients. In this study, we aimed to investigate
the associations of circulating GPC4 level with various factors in patients with
T2DM.
METHODS
This study included 152 patients with T2DM who were admitted to Jeju National
University Hospital from July 2010 to July 2013. The participants were part of the
Korea Diabetes Cohort Study, which was conducted after approval from the Jeju
National University Hospital Institutional Review Board (IRB No. 2010-06-033).
Written informed consent was obtained from all subjects in this study. The inclusion
criteria were patients with T2DM, aged with 18 to 80 years, who had given informed
consent. Exclusion criteria for our study were: type 1 diabetes mellitus (fasting
serum C-peptide <0.6 ng/mL and a history of diabetic ketoacidosis); patients
treated with a dipeptidyl peptidase-4 (DPP4) inhibitor, α-glucosidase
inhibitor, glucagon-like peptide-1 (GLP-1) agonist, or a thiazolidinedione, all
drugs known to affect plasma GLP-1 or gastric inhibitory polypeptide (GIP) level;
severe renal dysfunction (a glomerular filtration rate [GFR] less than 30
mL/min/1.73 m2); severe hepatic dysfunction (Child-Pugh score B or C);
and patients who were diabetic because of secondary causes.
We collected serum in plain tubes and plasma in ethylenediaminetetraacetic acid tubes
coated with aprotinin (25 µL/mL blood; Trasylol, SRL Inc., Tokyo, Japan) and
DPP4 inhibitor (10 µL/mL blood; Millipore, St. Charles, MO, USA) from the
enrolled patients to measure active GLP-1 and active GIP levels. All blood samples
were cooled on ice immediately and within 20 minutes of collection were centrifuged
at 4℃, after which serum/plasma was stored at -70℃ until
analysis.
Physical measurements (height, weight, waist circumference, and hip circumference),
blood pressure (BP), and biochemical laboratory test results were performed for all
participating patients. Waist circumference was measured twice at the narrowest part
between the chest and iliac crest parallel to the ground while maintaining normal
breathing. Measurements of insulin, C-peptide, glucose, glycated hemoglobin (HbA1c),
aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, blood
urea nitrogen, high-sensitivity C-reactive protein (hsCRP), total cholesterol, high
density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol
(LDL-C), triglycerides (TG), apolipoprotein A1 (ApoA1), and apolipoprotein B (ApoB)
were performed on blood samples obtained after 12 hours fasting. By interview or
reviewing medical records, we assessed the participants' histories of medication,
including antihypertensive medication, glucose-lowering agents, and lipid-lowering
agents, and the durations of their diabetes. We additionally measured adiponectin,
GPC4, and interleukin 6 (IL-6) in the collected plasma samples. We also measured
active GLP-1 and active GIP as biologically active forms in the collected plasma
samples.
GPC4 level was assayed using a commercially available ELI-SA kit (USCNK Life Science,
Houston, TX, USA), as were levels of adiponectin (Abcam, Cambridge, UK), IL-6
(Abcam), active GLP-1 (Immuno-Biological Laboratories, Takasaki, Japan), and active
GIP (Immuno-Biological Laboratories).
Analysis
The results are presented as mean±standard error of the mean. The
stratified quartile groups were analyzed using analysis of variance (ANOVA). We
used correlation analysis followed by linear regression analysis to identify the
factors that were independently associated with GPC4 level. All analyses were
performed with SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA), and
P<0.05 was defined as significant.
RESULTS
Baseline characteristics of T2DM patients
The mean age of the subjects in this study was 58.1 years, and 23.8% were women.
The subjects were mildly obese with an average body mass index (BMI) of 26.1
kg/m
2 and a waist-hip ratio of 1.0, with a mean disease duration
of 101.3 months. Their mean glucose status was 140.7 mg/dL fasting glucose and
7.5% HbA1c, and their mean insulin status was 8.4 µU/L of insulin, 2.2
ng/mL fasting C-peptide, 1.2 homeostatic model assessment of insulin resistance
(HOMA-IR), and 44.5% HOMA-β. They had reduced insulin resistance and
decreased ability for insulin secretion. The baseline characteristics of the
participants in this study are typical for Asian patients with T2DM (
Table 1). The average GPC4 concentration
was 2.0 ng/mL, that of adiponectin 2.8 µg/mL, and that of IL-6 9.2 pg/mL.
The levels of active GIP and GLP-1 were 3.2 and 5.2 pmol/L, respectively. Most
participants were being treated with metformin, and more than half of them with
sulfonylurea, lipid-lowering agents, and antihypertensive medications.
Factors associated with GPC4 level
The GPC4 level is known to differ between healthy men and women [
8]. Therefore, we compared the GPC4 level of
male participants with that of female participants (
Supplemental Table S1).
The GPC4 level did not differ between men and women with T2DM; nor did the level
of adiponectin, a surrogate marker of insulin sensitivity, or of IL-6, a marker
of inflammation. In the correlation analysis, adiponectin
(
r=0.005,
P=0.952) and IL-6
(
r=-0.083,
P=0.337) were not
significantly associated with GPC4 (data not shown).
Correlation analysis showed that GPC4 level in patients with T2DM was not
significantly correlated with any factor except age (
Fig. 1). No other factors, including fasting glucose, AST,
ALT, active GLP-1, BP, glucose-lowering agents, antihypertensive drugs, waist
circumference, smoking, disease duration, fasting insulin, C-peptide, HbA1c,
HbA1c quartile, BMI quartile, HOMA-β quartile, HOMA-β (%), hsCRP,
creatinine, estimated GFR, total cholesterol, TG, HDL-C, LDL-C, ApoB, ApoA1, the
ApoB/A1 ratio, and γ-glutamyl transpeptidase were significantly
associated with GPC4 in patients with T2DM.
We stratified the patients into quartiles based on BMI, HOMA-IR, and
HOMA-β (
Supplemental
Fig. 1S). The GPC4 did not differ significantly among quartile groups
stratified based on BMI. However, GPC4 level differed significantly among the
quartile groups stratified based on HOMA-IR and HOMA-β
(
P=0.040 and
P=0.049, respectively). By
linear regression, GPC4 also showed a decreasing trend in the higher HOMA-IR
quartiles (
P=0.033), but not in the HOMA-β quartiles
(
P=0.146).
Multivariate analysis of factors related to GPC4 level
The only factor in the correlation analysis that was significantly associated
with GPC4 level in T2DM patients was age. Therefore, multivariate analysis was
performed to identify factors independently associated with GPC4 by adjusting
for various factors. We included age, sex, and BMI as basic factors and added
variables with a
P<0.2 in the correlation analysis:
fasting glucose, AST, ALT, active GLP-1, and HOMA-IR quartile. We found that age
(β=0.224,
P=0.009), active GLP-1 (β=0.171,
P=0.049), and AST (β=-0.176,
P=0.043) were independently associated with GPC4 level
after adjusting for sex, BMI, fasting glucose, ALT, and HOMA-IR quartile (
Table 2).
DISCUSSION
In this study, we found that age and active GLP-1 level were positively associated
and AST was negatively associated with GPC4 level in patients with T2DM.
Previous studies have shown that GPC4 level was associated with insulin resistance
because it acts as an insulin sensitizer [
6,
8,
10]. In those studies, GPC4 was investigated in obese patients with or
without mild metabolic dysfunction such as impaired fasting glucose, impaired
glucose tolerance, or newly diagnosed diabetes. The GPC4 level was shown to be
increased in subjects with obesity, prediabetes, newly diagnosed diabetes, and fatty
liver disease compared with that in normal subjects. The GPC4 level was also
consistently positively correlated with a marker of obesity, such as the waist-hip
ratio, BMI, and fat distribution. Therefore, GPC4 was associated with insulin
resistance or metabolic disorders. The mechanisms of this association may originate
from the expression of GPC4 on cell membranes, especially in visceral fat, and the
release of circulating GPC4 from the cell surface by an enzymatically regulated
process mediated by the GPI lipase family [
6].
Obese subjects or those with fatty liver disease have higher insulin level, which
augments the activity of GPI-specific phospholipase D (GPLD-1), a member of the GPI
lipase family. This higher activity of GPLD-1 enzyme induced by higher insulin level
cleaves more GPC4 from cell surfaces, resulting in more circulating GPC4. Therefore,
elevated GPC4 in obesity is related to insulin resistance.
An animal study has shown that GPC4 was elevated in
ob/ob mice on a
high-fat diet that were normoglycemic and normoinsulinemic, whereas
ob/ob mice with elevated glucose and insulin levels showed
reduced GPC4 level [
6]. This indicates that
T2DM patients have different associations between GPC4 and other clinical factors
than do nondiabetic patients. In this study, we identified a correlation between
GPC4 and basal active GLP-1 in patients with T2DM. GLP-1 is an incretin hormone that
increases insulin secretion and reduces glucagon production in the pancreas. The
physiological role of GLP-1 was suggested to be the reduction of blood glucose level
after meals, but this proposal was based on analysis of the postprandial GLP-1
response, not of basal GLP-1 level [
11,
12]. In fact, the clinical meaning of basal
GLP-1 level is unclear. GLP-1 and GPC4 have similar effects in improving
hyperglycemia. In previous studies, GLP-1 responses were decreased in patients with
T2DM [
13], while another study suggested that
low GLP-1 level was associated with a risk of T2DM [
14], and a third study showed that basal active GLP-1 decreased in
patients with T2DM [
15]. A study in mice
found that GPC4 was elevated in normoglycemia mice on a high fat diet, but decreased
in mice with hyperglycemia [
6]. The fact that
active GLP-1 and GPC4 have similar effects may be why GPC4 and active GLP-1 are both
decreased in T2DM patients and are positively correlated with each other. Although
the mechanism behind this phenomenon should be investigated further, we demonstrated
an interesting association between GPC4 and basal active GLP-1 levels.
We found that GPC4 level in patients with T2DM was associated with age and AST level.
GPC4 level was higher in older patients than in younger patients; this could be
related to decreased renal function in older patients. Faerch et al. [
13] reported that GLP-1 responses were
positively correlated with age, milder obesity, and better insulin sensitivity. They
also suggested that the role of age in GLP-1 responses might be related to the
general reduction in renal clearance in older patients, which could explain the
higher basal active GLP-1 and GPC4 levels in older patients with T2DM seen in this
study. In addition, in this study, the AST level was negatively associated with GPC4
level in patients with T2DM. Yoo et al. [
8]
reported that GPC4 level was correlated with AST level and suggested that GPC4 level
was increased in women with NAFLD. We obtained the opposite result, showing a
negative correlation between GPC4 and AST in T2DM patients. The differences between
the two studies might be due to different subjects. Our patients had lower BMI and
lower insulin resistance than those in the study by Yoo et al. [
8], which might have affected the association
between GPC4 and AST levels. However, we showed that GPC4 was independently
associated with basal active GLP-1, age, and AST level, which suggested that GPC4
plays a different role in T2DM patients than that proposed previously.
In contrast, in this study, we found that increasing HOMA-IR quartile was negatively
associated with GPC4 level using ANO-VA, which is the opposite result to those of
previous studies [
6,
8,
10]. This phenomenon,
i.e., that the level of a hormone increases in prediabetes or metabolic syndrome and
decreases in diabetes mellitus, is also observed for other hormones. For example,
insulin level increases during the prediabetes and metabolic syndrome stages, but
decreases in full-blown T2DM. This decrease, together with the concomitant insulin
resistance, is involved in the pathophysiologic mechanisms of progress to T2DM
[
16]. The tendency for decreasing GPC4
level in the highest HOMA-IR quartile group might reflect the failure of patients
with T2DM to overcome insulin resistance. Alternatively, this difference might be
because the patients enrolled in this study were not typically obese T2DM patients,
but were only mildly obese, and did not have increased HOMA-IR, quite different
characteristics from Caucasian patients. These characteristics of patients in this
study might be causes to show a weak relationship between GPC4 and other disease
parameters, while previous studies showed other factors to be more strongly
associated with GPC4 level. These differences might have caused the different
results in the previous and this study.
In conclusion, active GLP-1 and AST levels were associated with GPC4 level in
patients with T2DM. Further experimental studies in models of T2DM are necessary to
clarify the mechanisms behind the correlation between GPC4 and active GLP-1 and the
role of GPC4 in patients with T2DM.
ACKNOWLEDGMENTS
This work was supported by the Academic Research Foundation of Jeju National
University Institute of Medical Science in 2014.
Supplementary Material
Supplemental Fig. S1
Comparisons by analysis of variance (ANOVA) of glypican-4 concentration
(ng/mL) in quartiles of patients stratified based on (A) body mass index
(BMI), (B) homeostasis model assessment of insulin resistance (HOMA-IR), and
(C) HOMA-β in patients with type 2 diabetes mellitus. The mean
HOMA-IR of the quartile groups were: 1st, 0.9±0.1; 2nd,
1.8±0.1; 3rd, 2.9±0.1; and 4th, 6.7±0.7
enm-31-439-s002.pdf
REFERENCES
1. Lillioja S, Mott DM, Spraul M, Ferraro R, Foley JE, Ravussin E, et al. Insulin resistance and insulin secretory dysfunction as
precursors of non-insulin-dependent diabetes mellitus. Prospective studies
of Pima Indians. N Engl J Med 1993;329:1988-1992.
[CROSSREF] [PUBMED]
2. Chen KW, Boyko EJ, Bergstrom RW, Leonetti DL, Newell-Morris L, Wahl PW, et al. Earlier appearance of impaired insulin secretion than of visceral
adiposity in the pathogenesis of NIDDM. 5-Year follow-up of initially
nondiabetic Japanese-American men. Diabetes Care 1995;18:747-753.
[CROSSREF] [PUBMED]
3. Hurt RT, Kulisek C, Buchanan LA, McClave SA. The obesity epidemic: challenges, health initiatives, and
implications for gastroenterologists. Gastroenterol Hepatol (N Y) 2010;6:780-792.
[PUBMED] [PMC]
4. Fico A, Maina F, Dono R. Fine-tuning of cell signaling by glypicans. Cell Mol Life Sci 2011;68:923-929.
[CROSSREF] [PUBMED] [PDF]
5. Gesta S, Bluher M, Yamamoto Y, Norris AW, Berndt J, Kralisch S, et al. Evidence for a role of developmental genes in the origin of
obesity and body fat distribution. Proc Natl Acad Sci U S A 2006;103:6676-6681.
[CROSSREF] [PUBMED] [PMC]
6. Ussar S, Bezy O, Bluher M, Kahn CR. Glypican-4 enhances insulin signaling via interaction with the
insulin receptor and serves as a novel adipokine. Diabetes 2012;61:2289-2298.
[CROSSREF] [PUBMED] [PMC]
7. Liu L, Gu H, Zhao Y, An L, Yang J. Glypican 4 may be involved in the adipose tissue redistribution
in high-fat feeding C57BL/6J mice with peroxisome proliferators-activated
receptor γ agonist rosiglitazone treatment. Exp Ther Med 2014;8:1813-1818.
[CROSSREF] [PUBMED] [PMC]
8. Yoo HJ, Hwang SY, Cho GJ, Hong HC, Choi HY, Hwang TG, et al. Association of glypican-4 with body fat distribution, insulin
resistance, and nonalcoholic fatty liver disease. J Clin Endocrinol Metab 2013;98:2897-2901.
[CROSSREF] [PUBMED] [PDF]
9. Li K, Xu X, Hu W, Li M, Yang M, Wang Y, et al. Glypican-4 is increased in human subjects with impaired glucose
tolerance and decreased in patients with newly diagnosed type 2
diabetes. Acta Diabetol 2014;51:981-990.
[CROSSREF] [PUBMED] [PDF]
10. Zhu HJ, Pan H, Cui Y, Wang XQ, Wang LJ, Li NS, et al. The changes of serum glypican4 in obese patients with different
glucose metabolism status. J Clin Endocrinol Metab 2014;99:E2697-E2701.
[CROSSREF] [PUBMED]
11. Laakso M, Zilinskaite J, Hansen T, Boesgaard TW, Vanttinen M, Stancakova A, et al. Insulin sensitivity, insulin release and glucagon-like peptide-1
levels in persons with impaired fasting glucose and/or impaired glucose
tolerance in the EU-GENE2 study. Diabetologia 2008;51:502-511.
[CROSSREF] [PUBMED] [PDF]
12. Smushkin G, Sathananthan A, Man CD, Zinsmeister AR, Camilleri M, Cobelli C, et al. Defects in GLP-1 response to an oral challenge do not play a
significant role in the pathogenesis of prediabetes. J Clin Endocrinol Metab 2012;97:589-598.
[CROSSREF] [PUBMED]
13. Faerch K, Torekov SS, Vistisen D, Johansen NB, Witte DR, Jonsson A, et al. GLP-1 response to oral glucose is reduced in prediabetes,
screen-detected type 2 diabetes, and obesity and influenced by sex: the
ADDITION-PRO study. Diabetes 2015;64:2513-2525.
[CROSSREF] [PUBMED]
14. Lastya A, Saraswati MR, Suastika K. The low level of glucagon-like peptide-1 (GLP-1) is a risk factor
of type 2 diabetes mellitus. BMC Res Notes 2014;7:849
[CROSSREF] [PUBMED] [PMC]
15. Toft-Nielsen MB, Damholt MB, Madsbad S, Hilsted LM, Hughes TE, Michelsen BK, et al. Determinants of the impaired secretion of glucagon-like peptide-1
in type 2 diabetic patients. J Clin Endocrinol Metab 2001;86:3717-3723.
[CROSSREF] [PUBMED]
16. Nathan DM. Clinical practice. Initial management of glycemia in type 2
diabetes mellitus. N Engl J Med 2002;347:1342-1349.
[CROSSREF] [PUBMED]
Fig. 1
Correlation curves of plasma glypican-4 concentration with (A) age, (B)
active glucagon-like protein 1 (GLP-1), (C) fasting glucose, and (D) aspartate
aminotransferase (AST) in patients with type 2 diabetes mellitus. Spearman
correlation coefficients and corresponding P values are
displayed.
Table 1
Baseline Characteristics of Patients
Factor |
Value |
Age, yr |
58.1±0.7 |
Female sex, % |
23.8 |
Body mass index,
kg/m2
|
26.1±0.3 |
Waist hip ratio |
1.0±0.0 |
Systolic blood
pressure, mm Hg |
142.5±1.4 |
Diastolic blood pressure,
mm Hg |
84.0±0.9 |
DM duration,
mo |
101.3±7.1 |
Glucose, mg/dL |
140.7±2.9 |
Insulin,
μU/mL |
8.4±0.5 |
C-peptide, ng/mL |
2.2±0.1 |
HbA1c, % |
7.5±0.1 |
HOMA-IR |
1.2±0.1 |
HOMA-β,
% |
44.5±2.4 |
hsCRP, mg/dL |
0.2±0.0 |
Blood urea
nitrogen, mg/dL |
16.4±0.4 |
Creatinine, mg/dL |
1.1±0.0 |
GFR, mL/min/1.73
m2
|
71.2±0.8 |
Total cholesterol,
mg/dL |
167.6±2.7 |
Triglyceride,
mg/dL |
133.7±6.6 |
HDL-C, mg/dL |
48.1±1.0 |
LDL-C, mg/dL |
99.5±2.5 |
AST, IU/L |
27.1±1.22 |
ALT, IU/L |
33.1±1.9 |
γGT, IU/L |
32.3±1.6 |
GLP-1, pmol/L |
5.2±0.3 |
GIP, pmol/L |
3.2±0.3 |
Glypican-4,
ng/mL |
2.0±0.2 |
Adiponectin,
μg/mL |
2.8±0.0 |
IL-6, pg/mL |
9.2±2.5 |
Sulfonylurea, % |
58.7 |
Metformin, % |
77.4 |
Insulin, % |
15.5 |
Statin, % |
51.0 |
Antihypertensive
medication, % |
52.3 |
Table 2
Multivariate Analysis to Identify Factors Associated with Glypican-4
Factor |
β |
P value |
Age, yr |
0.224 |
0.009 |
Female sex |
-0.015 |
0.862 |
BMI,
kg/m2
|
-0.057 |
0.515 |
Glucose, mg/dL |
-0.097 |
0.258 |
HOMA-IR
(quartiles) |
-0.123 |
0.185 |
AST, IU/L |
-0.176 |
0.043 |
ALT, IU/L |
-0.040 |
0.779 |
Active GLP-1, pmol/L |
0.171 |
0.049 |