European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 98–101
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Serum vaspin levels and vaspin mRNA expression in subcutaneous adipose tissue in women with gestational diabetes mellitus Wei Qian Mm, Jianxia Fan *, Shuzin Khor, Mengfan Song, Wei Hong, Xiaobei Dai Department of Gynecology and Obstetrics, Shanghai Jiao Tong University, International Peace Maternity and Child Health Hospital, 910 Hengshan Road, Shanghai 200030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 June 2014 Received in revised form 25 August 2014 Accepted 3 September 2014
Objective: To compare serum vaspin level and mRNA and protein levels of vaspin in adipose tissue in women with gestational diabetes mellitus (GDM) and normal glucose tolerance (NGR), along with the correlation between serum vaspin level with fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR) and birth-weight. Study design: Thirty-seven women with GDM and 36 with NGR were enrolled. Total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting plasma glucose (FPG), FINS and vaspin levels were measured. The mRNA and protein levels were detected using RT-PCR and Western blot. Pearson correlation analysis (PCA) was performed to reveal the correlation between serum vaspin level and FINS, HOMA-IR. Spearman correlation analysis (SCA) was conducted to examine the association between serum vaspin level and birth-weight. Results: HDL-C level in GDM was lower than NGR group (P < 0.05), and there were no statistical differences in TC, TG, LDL-C, FPG, FINS and HOMA-IR between the two groups. Serum vaspin level, mRNA and protein expression levels of vaspin in GDM were higher than NGR group (P < 0.05). Serum vaspin level was not significantly correlated with FINS and HOMA-IR, but had a positive correlation with birthweight (P = 0.023). Conclusion: Serum vaspin level cannot serve as an independent predictor of IR. The increased serum vaspin level and increased vaspin mRNA and protein expression in adipose tissues in GDM women indicate that vaspin may be involved in the pathogenesis of GDM, but its exact mechanism needs further study. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Serum vaspin Vaspin mRNA Subcutaneous adipose tissue Gestational diabetes mellitus
Introduction Human pregnancy confers a state of insulin resistance (IR) and hyperinsulinemia that may predispose some women to diabetes. As pregnancy advances, IR becomes more and more intense and gestational diabetes mellitus (GDM) may occur when a woman’s pancreatic function is not sufficient to overcome the diabetogenic environment of pregnancy [1]. The majority of GDM manifests at the 24–28th week of gestation [2]. Traditional studies suggest that GDM is resulted from the effects of placental hormones [3]. In recent years, a large amount of evidence has supported the role of adipose tissue in the regulation of IR in GDM
* Corresponding author. Tel.: +86 021 64070434; fax: +86 21 64073421. E-mail address: [email protected] (J. Fan). http://dx.doi.org/10.1016/j.ejogrb.2014.09.008 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
[4]. Therefore, some adipocytokines have been shown to be correlated with maternal metabolism and gestational IR. Visceral adipose tissue-derived serine protease inhibitor (vaspin), a member of serine enzyme inhibitor family, has been identified in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat in 2005 by Hida et al. [5]. Many researches have been devoted to vaspin since it was identified [6–10]. It is an adipokine which might be involved in glucose regulation by increasing the insulin sensitivity [11]. It has been reported that there was a higher vaspin mRNA expression in adipose tissues of obesity and type 2 diabetes mellitus (T2DM) patients, and this was considered as a compensatory mechanism for severe IR [12,13]. The circulating vaspin level is likely to reflect its expression in adipose tissues [7]. To date, the correlation between circulating vaspin level and the development of GDM was controversial. It has been reported that serum vaspin level was negatively correlated with serum adiponectin level in obese people [14], and was positively correlated with body mass index
W.Q. Mm et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 98–101
(BMI), triglycerides (TG) and IR in several studies [7,15,16]. However, other studies hold different opinions. Stepan et al. suggest that serum vaspin level was not significantly different between GDM and non-GDM women, and serum vaspin level had no significant correlation with IR [17]. A recent report further suggest that vaspin level was positively correlated to IR and TG at the 3rd trimester of pregnancy but these correlations were not observed at the 2nd trimester or postpartum [18]. This prospective study aimed to determine serum vaspin level and the mRNA and protein expression level of vaspin in subcutaneous adipose tissue of GDM and non-GDM women. Besides, it was to evaluate the correlation between serum vaspin levels, HOMA-IR and birth-weight in GDM women, and to further explore the role of vaspin in the pathogenesis of GDM.
Materials and methods This study was approved by the ethics committee of our hospital and was in accordance with the Declaration of Helsinki. Written informed consents were obtained from all participants. Patients Seventy-three pregnant women who underwent complete antenatal examination in Shanghai Jiaotong University affiliated to International Peace Maternity and Child Health Hospital from January 2012 to October 2012 were selected. Selected demographic and clinical details of pregnant women who had regular inspection in our department in the whole year of 2012 were recorded. The details included ages, height, pre-pregnant weight, gravidity, gestational age, etc. According to the results of oral glucose tolerance test (OGTT) at 24–28th week (the standard was described below), 37 GDM women were selected as the experimental group and 36 pregnant women with normal glucose tolerance were taken as the control group. The ages, gestational ages, gravidity and BMI of control group were all matched to the experimental group. Each group was then divided into two subgroups (elevated BMI (EBMI) or normal weight (NW)) according to their pre-pregnancy BMI. The inclusion criteria were as follows: (a) all of them aged 31.7 4.7 year; (b) all of them had one or two gravidity; (c) gestational age was 273.8 5.7 day, which was determined by obstetricians considering patients’ dates, clinical assessments and ultrasound examination; (d) BMI was 26.1 4.5 kg/ m2 which was determined by dividing the pre-pregnancy weight (in kilograms) by recall by the height (in meters) squared. The pregnant women who had the following situations were excluded from both groups: (1) type I or II diabetes before pregnancy; (2) hypertensive disorders in pregnancy; (3)severe liver, kidney and cardiovascular disease; and (4) any medication that would affect glucolipid metabolism during pregnancy, such as corticosteroids. Diagnosis for GDM and the indications for cesarean section According to the standard recommended by International Association of Diabetes and Pregnancy Study Groups (IADPSG) [19], an oral glucose tolerance test (OGTT) using 75 g glucose load was performed on all the enrolled women, besides, fasting, 1 h and 2 h samples were recorded. GDM diagnosis [20–22] could be diagnosed if one or more values met or exceeded the following levels of glucose: fasting 5.1 mmol/l, 1 h post glucose 10.0 mmol/l and 2 h post glucose 8.5 mmol/l. The indications for cesarean section includes: breech presentation, scarred uterus (after myoma enucleation), fetal macrosomia, advanced maternal age (>35 years old) and the patient’s own request even though they did not have any indications for cesarean section.
99
Fasting blood draw and data collection At the early morning of the day when cesarean section was performed, 8 ml fasting blood samples were taken from all women. The specimen was centrifuged at 3000 r/min for 10 min and separated to three serum samples. One sample was used for determination of total cholesterol (TC), TG, high density lipoproteincholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and FPG. The other two samples were stored at 80 8C for separate measurement of fasting insulin (FINS) and vaspin levels. Serum FINS was assayed with commercially available radioimmunoassay kits (Beckman Instruments, Inc., Fullerton, CA). Commercial enzymelinked immunosorbent assay (ELISA kits, ALPCO, Diagnostics, Windham, NH, USA–linearity range 0.016–1 ng/ml, intra-assay coefficient of variation (CV) < 5%, inter-assay CV < 10%)) were used to measure serum vaspin. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated according to the formula: FINS (mU/L) FPG (nmol/L)/22.5 [23]. A 1 cm3 subcutaneous adipose tissue block was taken during cesarean section surgery, and was fast transferred to a 80 8C refrigerator. The above processes were performed under strict aseptic conditions. The mRNA expression was detected by reverse transcription-polymerase chain reaction technology (RT-PCR) (TaKaRa PrimeScriptTM RT-PCR Kit; TaKaRa, Japan). Besides, protein expression levels of vaspin were determined by Western blotting (Oxford Biomedical Research, Oxford, MI) using a human vaspin-specific monoclonal antibody (AdipoGen; Inc.; Seoul, South Korea). Statistical analysis Measurement data were expressed as mean standard deviation. T test was used for comparison within the group and between groups. Pearson correlation analysis (PCA) was used to analyze the correlation between serum vaspin level and IR. Spearman correlation analysis (SCA) was conducted to examine the association between serum vaspin level and birth-weight. SPSS software version 13.0 (SPSS, Chicago, IL) was used, and P < 0.05 was considered statistically significant. Results The general information of all pregnant women was shown in Table 1. Thirty-seven cases of pregnant women with abnormal glucose tolerance were included into the GDM group, and 36 cases of pregnant women with normal glucose tolerance (NGR) were included into NGR group. Subsequently, each group was divided into two subgroups of EBMI and NW according to pre-pregnant BMI. Seventeen cases in the GDM group were included into GDMEBMI subgroup (pre-pregnancy BMI 25 kg/m2) and 20 cases were included into GDM-NW sub-group (pre-pregnancy BMI < 25 kg/m2). The control group consisted of 20 cases in NGR-EBMI subgroup and 16 cases in NGR-NW sub-group. Comparisons of the glucolipid metabolism, HOMA-IR and serum vaspin levels between GDM and NGR groups were shown in Table 2. The HDL-C level in NGR group were significantly higher Table 1 Comparison of general condition between the two groups. Factor
N Age (year) Gravidity (median-range) Gestational age(day) BMI
NGR
GDM
NGR-NW
NGR-EBMI GDM-NW
EBMI-GDM
16 31.8 3.4 1 (1–1) 272.6 3.8 22.6 1.0
20 31.3 4.3 1 (1–1) 274.0 3.6 27.7 2.9
19 32.1 3.7 1 (1–2) 272.7 6.8 27.8 2.4
18 32.4 4.0 1 (1–1) 271.9 3.8 23.2 0.9
NGR: normal glucose tolerance; GDM: gestational diabetes mellitus; NW: normal weight; BMI: body mass index; EBMI: elevated BMI.
W.Q. Mm et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 98–101
100
than those in GDM group (P = 0.0095). While, the serum vaspin level in NGR group were significantly lower than those in GDM group (P = 0.0012). However, there were no statistical differences in TC, TG, LDL-C, FPG, FINS and HOMA-IR between these two groups (P > 0.05). A comparison was carried out within the GDM group, and result showed that there were no statistical differences in all indexes of glucolipid metabolism, HOMA-IR and serum vaspin level between subgroups (P > 0.05). However, TC, FPG, FINS and HOMA-IR in NGR-EBMI subgroup were significantly higher than those in NGR-NW subgroup (P < 0.05). Vaspin mRNA expression level in subcutaneous adipose tissue in the GDM group was significantly higher than that in the NGR group (P = 0.0003; Fig. 1). In addition, there was significant difference in the two groups (NW-NGR versus EBMI-NGR, P = 0.000; NW-GDM versus EBMI-GDM, P = 0.000). However, there was no significance in EBMI-NGR and EBMI-GDM (P = 0.086). Similarly, protein expression levels of vaspin in subcutaneous adipose tissues in the GDM group presented higher level than that in the NGR group (P = 0.025). Moreover, there were also significant differences in subgroups of GDM and NGR (NW-NGR versus EBMINGR, P = 0.016; NW-GDM versus EBMI-GDM, P = 0.035). PCA indicated that serum vaspin level was not significantly correlated with FINS and HOMA-IR in GDM women in all groups; however, there was a significant positive correlation between serum vaspin level and birth-weight (r = 0.266, P = 0.023). Comments As a recently discovered adipokine, the specific mechanism of vaspin is not yet fully determined. Current studies mostly focus on T2DM and obese patients, and the vaspin is considered as an insulin sensitizer which may be involved in regulation of endogenous glucose metabolism. Recently, more and more researchers began to study the roles of vaspin in GDM. In this study, we evaluated the serum vaspin levels and mRNA and protein expression levels of vaspin in subcutaneous adipose tissue in pregnant women with GDM or NGR. Our results suggested that vaspin levels in both serum and adipose tissue were significantly higher in GDM than those in NGR, however, serum vaspin level was not significantly correlated with FINS and HOMA-IR in GDM women, but had a positive correlation with birth-weight. Vaspin, a secretory protein, is mainly synthesized by the visceral white adipose tissue [24]. Using enzyme linked immunosorbent assay (ELISA), Western blot and RT-PCR techniques, Tan et al. [25] found that patients with polycystic ovary syndrome (PCOS) have increased serum vaspin levels and higher vaspin mRNA expression levels in omental adipose tissues. The same findings were revealed in our study. The vaspin mRNA and protein
Fig. 1. Comparison of vaspin mRNA expression levels in subcutaneous adipose tissue between the four subgroups. NW-NGR represents normal weight with normal glucose tolerance; EBMI-NGR represents elevated BMI with normal glucose tolerance; GDM represents gestational diabetes mellitus; NW-GDM represents normal weight with gestational diabetes mellitus, EBMI-GDM represents elevated BMI with gestational diabetes mellitus. BMI represents body mass index; # represents P < 0.05 compared to other three groups; * represents P < 0.001 compared to other three groups.
expressions in subcutaneous adipose tissues of GDM patients were significantly higher than those in the NGR group, and correspondingly, the serum vaspin levels in GDM patients were also significantly higher than those in the NGR group. Both GDM and PCOS patients have insulin resistance and would develop T2DM in a few years (pre-diabetic state). Moreover, PCOS women during pregnancy are also more prone to develop GDM. Therefore, we hypothesized that their pathogenesis might have some similarity. However, further studies are needed to confirm this hypothesis. In the non-pregnant state, elevated blood glucose levels are positively correlated with elevated serum vaspin levels in obese or diabetic patients [7], and serum vaspin levels are significantly decreased after metformin treatment [26]. Blood glucose and serum vaspin levels are lower, and serum insulin levels are higher in pregnant women compared with non-pregnant women [4]. Our results showed that, in late pregnancy, serum vaspin levels in GDM women were higher than those in the NGR group, but were not significantly correlated with serum FINS and HOMA-IR. This was consistent with an in vitro experimental result which demonstrated that vaspin levels were increased by adding glucoses in the adipose tissues, while not changed when adding insulin [25]. Stepan et al. found that there was no significant difference in serum vaspin level between GDM and non-GDM women, and the
Table 2 Comparison of the glucolipid metabolism and serum vaspin levels between the two groups. Factor
TC (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) FPG (mmol/l) FINS (mU/L) HOMA-IR Vaspin (ng/ml) *
NGR
GDM
NGR-NW
NGR-EBMI
GDM-NW
GDM-EBMI
P (inter-groups)
6.3 0.9 2.9 1.1 1.9 0.3 4.0 0.9 3.4 1.0 6.2 3.1 1.0 0.6 1.3 0.7
5.7 0.8* 3.2 0.9 1.7 0.3 3.7 0.7 4.2 1.3* 15. 8 12.7** 3. 5 4.7* 1.6 0.6
5.5 1.0 3.1 1.0 1.6 0.3 3.4 0.7 4.4 1.1 7.5 5.6 1.6 1.4 2.2 1.8
6.0 1.1 3.6 1.0 1.7 0.3 3.8 0.9 4.0 0.5 7.6 3.8 1.4 0.7 3.12 2.1
0.196 0.168 0.0095 0.1508 0.1047 0.0726 0.2264 0.0012
P < 0.05 for comparison within NGR or GDM group. P < 0.001 for comparison within NGR or GDM group. NGR: normal glucose tolerance; GDM: gestational diabetes mellitus; NW: normal weight; EBMI: elevated BMI; TC: total cholesterol; TG: triglyceride; HDL-C: high density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; FPG: fasting plasma glucose; FINS: fasting insulin; HOMA-IR: homeostasis model assessment of insulin resistance. **
W.Q. Mm et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 98–101
serum vaspin level had no significant correlation with insulin sensitivity in pregnant subjects [17]. However, in our study, the serum vaspin level in GDM group was significantly higher than that in non-GDM women. We hypothesized that this might result from the elimination of influence of EBMI in our study. In the study, the GDM and NGR groups were respectively divided into subgroups according to pre-pregnancy BMI, and BMI were matched between GDM-EBMI and NGR-EBMI subgroups. In addition, we know that in late pregnancy, due to the influence of factors such as fetal weight and amniotic fluid volume, there would be a relatively large error in measuring BMI and waist–hip ratio (WHR), thus the pre-pregnancy BMI were used for grouping in this study. Furthermore, it might be considered as the compensative increase of vaspin in order to ameliorate the increased IR in GDM. Besides, this study suggested that serum vaspin level had no significant correlation with glucolipid metabolism and insulin sensitivity in pregnant women, but there were significant differences in TC, FPG, FINS and HOMA-IR in NGR-EBMI and in NGR-NW subgroup. The former result was consistent with that of Stepan et al. It was, therefore, reasonable to speculate that the serum vaspin level cannot be used as an independent predictor of IR. The latter result indicated that the glucolipid metabolism was related to EBMI. However, the serum vaspin level shows a positive correlation with IR in the non-pregnant population of diabetes and obesity [7,14]. This is due to the fact that the occurrence of IR in pregnant women is very complex. In addition to adipose tissue, it has been well demonstrated that vaspin is also expressed in human and rat placenta. Besides, vaspin increases gradually during pregnancy, and elevates to the highest level at the end of gestation [27].Therefore, the placenta cannot be ignored owing to its effect of endocrine. Moreover, it has a considerable influence on glucolipid metabolism and IR during gestation period. Furthermore, we also found that there was a positive correlation between serum vaspin level and birth-weight. A previous study suggested that a positive correlation between maternal and fetal vaspin concentrations, which implied a transplacental transport of adipocytokines and speculated that vaspin may derive from fetal and/or maternal tissues [28]. In addition, other studies have recently indicated that the mRNA of vaspin level in adipose tissue is related to obesity and glucose metabolism [7,13]; therefore, we suspected that the birth-weight of infants may be indirectly influenced by maternal vaspin level. However, in this study, we did not measure vaspin mRNA and protein expression levels in placental tissues and infants, therefore it was unable to assess their roles in IR, and the regulatory mechanism (e.g. birth-weight of infants) for serum vaspin level was also not very clear. In summary, the serum vaspin levels do not reflect the degrees of IR in GDM women, that is to say, the serum vaspin level cannot serve as an independent predictor of IR. However, the serum vaspin level increases with mRNA expression level of the vaspin in subcutaneous adipose tissues in GDM women, indicating that the vaspin may be involved in the pathogenesis of GDM. In view of the relatively complex etiology of GDM, we only know that the vaspin might be involved in the occurrence of GDM, but its specific regulatory mechanism needs further study. Conflict of interest All authors declare that there is no conflict of interest in this study. Condensation Serum vaspin level is significantly higher in GDM group than those in NGR groupand is not significantly correlated with FINS and HOMA-IR.
101
References [1] Gilmartin AB, Ural SH, Repke JT. Gestational diabetes mellitus. Rev Obstet Gynecol 2008;1(3 (Summer)):129–34. [2] Butte NF. Carbohydrate and lipid metabolism in pregnancy: normal compared with gestational diabetes mellitus. Am J Clin Nutr 2000;71(5 Suppl (May)): S61–1256. [3] Briana DD, Malamitsi-Puchner A. Reviews: adipocytokines in normal and complicated pregnancies. Reprod Sci 2009;16(10 (Oct)):921–37. [4] Giomisi A, Kourtis A, Toulis KA, et al. Serum vaspin levels in normal pregnancy in comparison with non-pregnant women. Eur J Endocrinol 2011;164(4): 579–83. [5] Hida K, Wada J, Eguchi J, et al. Visceral adipose tissue-derived serine protease inhibitor: a unique insulin-sensitizing adipocytokine in obesity. Proc Natl Acad Sci USA 2005;102(30):10610–15. [6] Bluher M. Vaspin in obesity and diabetes: pathophysiological and clinical significance. Endocrine 2012;(2):176–82. [7] Youn BS, Kloting N, Kratzsch J, et al. Serum vaspin concentrations in human obesity and type 2 diabetes. Diabetes 2008;57(2):372–7. [8] Li Z, Ma C, Li L, Pan X, Chen L. Vaspin serum concentration in patients with type 2 diabetes and carotid plaque. J Int Med Res 2012;40(5):1670–6. [9] Esteghamati A, Noshad S, Mousavizadeh M, Zandieh A, Nakhjavani M. Response: association of vaspin with metabolic syndrome: the pivotal role of insulin resistance. Diabetes Metab J 2014;38(3):242–3. [10] Jia X, Wang S, Ma N, et al. Comparative analysis of vaspin in pregnant women with and without gestational diabetes mellitus and healthy non-pregnant women. Endocrine 2014;15 [Epub ahead of print]. [11] Aust G, Richter O, Rohm S, et al. Vaspin serum concentrations in patients with carotid stenosis. Atherosclerosis 2009;204(1):262–6. [12] Zvonic S, Lefevre M, Kilroy G, et al. Secretome of primary cultures of human adipose-derived stem cells: modulation of serpins by adipogenesis. Mol Cell Proteomics 2007;6(1):18–28. [13] Kloting N, Berndt J, Kralisch S, et al. Vaspin gene expression in human adipose tissue: association with obesity and type 2 diabetes. Biochem Biophys Res Commun 2006;339(1):430–6. [14] Suleymanoglu S, Tascilar E, Pirgon O, Tapan S, Meral C, Abaci A. Vaspin and its correlation with insulin sensitivity indices in obese children. Diabetes Res Clin Pract 2009;84(3):325–8. [15] Teshigawara S, Wada J, Hida K, et al. Serum vaspin concentrations are closely related to insulin resistance, and rs77060950 at SERPINA12 genetically defines distinct group with higher serum levels in Japanese population. J Clin Endocrinol Metab 2012;97(7 (Jul)):E1202–07. [16] Handisurya A, Riedl M, Vila G, et al. Serum vaspin concentrations in relation to insulin sensitivity following RYGB-induced weight loss. Obes Surg 2010;20(2 (Feb)): 198–203. [17] Stepan H, Kralisch S, Klostermann K, et al. Preliminary report: circulating levels of the adipokine vaspin in gestational diabetes mellitus and preeclampsia. Metabolism 2010;59(7):1054–6. [18] Gkiomisi A, Makedou KG, Anastasilakis AD, et al. Serum vaspin levels in women with and without gestational diabetes mellitus during pregnancy and postpartum. Cytokine 2013;61(1 (Jan)):127–32. [19] Metzger BE, Gabbe SG, Persson B, et al. International association of diabetes and pregnancy study groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 2010;33(3): 676–82. [20] Metzger BE, Coustan DR. Summary and recommendations of the Fourth International Workshop-Conference on gestational diabetes mellitus. The Organizing Committee. Diabetes Care 1998;21(Suppl 2 (Aug)): B161–7. [21] Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. National Diabetes Data Group. Diabetes 1979;28(12): 1039–57. [22] Carpenter MW, Coustan DR. Criteria for screening tests for gestational diabetes. Am J Obstet Gynecol 1982;144(7):768–73. [23] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28(7):412–9. [24] Hida K, Wada J, Zhang H, et al. Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats. J Lipid Res 2000;41(10): 1615–22. [25] Tan BK, Heutling D, Chen J, et al. Metformin decreases the adipokine vaspin in overweight women with polycystic ovary syndrome concomitant with improvement in insulin sensitivity and a decrease in insulin resistance. Diabetes 2008;57(6):1501–7. [26] Gulcelik NE, Karakaya J, Gedik A, Usman A, Gurlek A. Serum vaspin levels in type 2 diabetic women in relation to microvascular complications. Eur J Endocrinol 2009;160(1):65–70. [27] Caminos JE, Bravo SB, Garces MF, et al. Vaspin and amylin are expressed in human and rat placenta and regulated by nutritional status. Histol Histopathol 2009;24(8):979–90. [28] Briana DD, Boutsikou M, Baka S, et al. Omentin-1 and vaspin are present in the fetus and neonate, and perinatal concentrations are similar in normal and growth-restricted pregnancies. Metab Clin Exp 2011;60(4): 486–90.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Serum vaspin levels and vaspin mRNA expression in subcutaneous adipose tissue in women with gestational diabetes mellitus Wei Qian Mm, Jianxia Fan *, Shuzin Khor, Mengfan Song, Wei Hong, Xiaobei Dai Department of Gynecology and Obstetrics, Shanghai Jiao Tong University, International Peace Maternity and Child Health Hospital, 910 Hengshan Road, Shanghai 200030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 June 2014 Received in revised form 25 August 2014 Accepted 3 September 2014
Objective: To compare serum vaspin level and mRNA and protein levels of vaspin in adipose tissue in women with gestational diabetes mellitus (GDM) and normal glucose tolerance (NGR), along with the correlation between serum vaspin level with fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR) and birth-weight. Study design: Thirty-seven women with GDM and 36 with NGR were enrolled. Total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting plasma glucose (FPG), FINS and vaspin levels were measured. The mRNA and protein levels were detected using RT-PCR and Western blot. Pearson correlation analysis (PCA) was performed to reveal the correlation between serum vaspin level and FINS, HOMA-IR. Spearman correlation analysis (SCA) was conducted to examine the association between serum vaspin level and birth-weight. Results: HDL-C level in GDM was lower than NGR group (P < 0.05), and there were no statistical differences in TC, TG, LDL-C, FPG, FINS and HOMA-IR between the two groups. Serum vaspin level, mRNA and protein expression levels of vaspin in GDM were higher than NGR group (P < 0.05). Serum vaspin level was not significantly correlated with FINS and HOMA-IR, but had a positive correlation with birthweight (P = 0.023). Conclusion: Serum vaspin level cannot serve as an independent predictor of IR. The increased serum vaspin level and increased vaspin mRNA and protein expression in adipose tissues in GDM women indicate that vaspin may be involved in the pathogenesis of GDM, but its exact mechanism needs further study. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Serum vaspin Vaspin mRNA Subcutaneous adipose tissue Gestational diabetes mellitus
Introduction Human pregnancy confers a state of insulin resistance (IR) and hyperinsulinemia that may predispose some women to diabetes. As pregnancy advances, IR becomes more and more intense and gestational diabetes mellitus (GDM) may occur when a woman’s pancreatic function is not sufficient to overcome the diabetogenic environment of pregnancy [1]. The majority of GDM manifests at the 24–28th week of gestation [2]. Traditional studies suggest that GDM is resulted from the effects of placental hormones [3]. In recent years, a large amount of evidence has supported the role of adipose tissue in the regulation of IR in GDM
* Corresponding author. Tel.: +86 021 64070434; fax: +86 21 64073421. E-mail address: [email protected] (J. Fan). http://dx.doi.org/10.1016/j.ejogrb.2014.09.008 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
[4]. Therefore, some adipocytokines have been shown to be correlated with maternal metabolism and gestational IR. Visceral adipose tissue-derived serine protease inhibitor (vaspin), a member of serine enzyme inhibitor family, has been identified in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat in 2005 by Hida et al. [5]. Many researches have been devoted to vaspin since it was identified [6–10]. It is an adipokine which might be involved in glucose regulation by increasing the insulin sensitivity [11]. It has been reported that there was a higher vaspin mRNA expression in adipose tissues of obesity and type 2 diabetes mellitus (T2DM) patients, and this was considered as a compensatory mechanism for severe IR [12,13]. The circulating vaspin level is likely to reflect its expression in adipose tissues [7]. To date, the correlation between circulating vaspin level and the development of GDM was controversial. It has been reported that serum vaspin level was negatively correlated with serum adiponectin level in obese people [14], and was positively correlated with body mass index
W.Q. Mm et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 98–101
(BMI), triglycerides (TG) and IR in several studies [7,15,16]. However, other studies hold different opinions. Stepan et al. suggest that serum vaspin level was not significantly different between GDM and non-GDM women, and serum vaspin level had no significant correlation with IR [17]. A recent report further suggest that vaspin level was positively correlated to IR and TG at the 3rd trimester of pregnancy but these correlations were not observed at the 2nd trimester or postpartum [18]. This prospective study aimed to determine serum vaspin level and the mRNA and protein expression level of vaspin in subcutaneous adipose tissue of GDM and non-GDM women. Besides, it was to evaluate the correlation between serum vaspin levels, HOMA-IR and birth-weight in GDM women, and to further explore the role of vaspin in the pathogenesis of GDM.
Materials and methods This study was approved by the ethics committee of our hospital and was in accordance with the Declaration of Helsinki. Written informed consents were obtained from all participants. Patients Seventy-three pregnant women who underwent complete antenatal examination in Shanghai Jiaotong University affiliated to International Peace Maternity and Child Health Hospital from January 2012 to October 2012 were selected. Selected demographic and clinical details of pregnant women who had regular inspection in our department in the whole year of 2012 were recorded. The details included ages, height, pre-pregnant weight, gravidity, gestational age, etc. According to the results of oral glucose tolerance test (OGTT) at 24–28th week (the standard was described below), 37 GDM women were selected as the experimental group and 36 pregnant women with normal glucose tolerance were taken as the control group. The ages, gestational ages, gravidity and BMI of control group were all matched to the experimental group. Each group was then divided into two subgroups (elevated BMI (EBMI) or normal weight (NW)) according to their pre-pregnancy BMI. The inclusion criteria were as follows: (a) all of them aged 31.7 4.7 year; (b) all of them had one or two gravidity; (c) gestational age was 273.8 5.7 day, which was determined by obstetricians considering patients’ dates, clinical assessments and ultrasound examination; (d) BMI was 26.1 4.5 kg/ m2 which was determined by dividing the pre-pregnancy weight (in kilograms) by recall by the height (in meters) squared. The pregnant women who had the following situations were excluded from both groups: (1) type I or II diabetes before pregnancy; (2) hypertensive disorders in pregnancy; (3)severe liver, kidney and cardiovascular disease; and (4) any medication that would affect glucolipid metabolism during pregnancy, such as corticosteroids. Diagnosis for GDM and the indications for cesarean section According to the standard recommended by International Association of Diabetes and Pregnancy Study Groups (IADPSG) [19], an oral glucose tolerance test (OGTT) using 75 g glucose load was performed on all the enrolled women, besides, fasting, 1 h and 2 h samples were recorded. GDM diagnosis [20–22] could be diagnosed if one or more values met or exceeded the following levels of glucose: fasting 5.1 mmol/l, 1 h post glucose 10.0 mmol/l and 2 h post glucose 8.5 mmol/l. The indications for cesarean section includes: breech presentation, scarred uterus (after myoma enucleation), fetal macrosomia, advanced maternal age (>35 years old) and the patient’s own request even though they did not have any indications for cesarean section.
99
Fasting blood draw and data collection At the early morning of the day when cesarean section was performed, 8 ml fasting blood samples were taken from all women. The specimen was centrifuged at 3000 r/min for 10 min and separated to three serum samples. One sample was used for determination of total cholesterol (TC), TG, high density lipoproteincholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and FPG. The other two samples were stored at 80 8C for separate measurement of fasting insulin (FINS) and vaspin levels. Serum FINS was assayed with commercially available radioimmunoassay kits (Beckman Instruments, Inc., Fullerton, CA). Commercial enzymelinked immunosorbent assay (ELISA kits, ALPCO, Diagnostics, Windham, NH, USA–linearity range 0.016–1 ng/ml, intra-assay coefficient of variation (CV) < 5%, inter-assay CV < 10%)) were used to measure serum vaspin. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated according to the formula: FINS (mU/L) FPG (nmol/L)/22.5 [23]. A 1 cm3 subcutaneous adipose tissue block was taken during cesarean section surgery, and was fast transferred to a 80 8C refrigerator. The above processes were performed under strict aseptic conditions. The mRNA expression was detected by reverse transcription-polymerase chain reaction technology (RT-PCR) (TaKaRa PrimeScriptTM RT-PCR Kit; TaKaRa, Japan). Besides, protein expression levels of vaspin were determined by Western blotting (Oxford Biomedical Research, Oxford, MI) using a human vaspin-specific monoclonal antibody (AdipoGen; Inc.; Seoul, South Korea). Statistical analysis Measurement data were expressed as mean standard deviation. T test was used for comparison within the group and between groups. Pearson correlation analysis (PCA) was used to analyze the correlation between serum vaspin level and IR. Spearman correlation analysis (SCA) was conducted to examine the association between serum vaspin level and birth-weight. SPSS software version 13.0 (SPSS, Chicago, IL) was used, and P < 0.05 was considered statistically significant. Results The general information of all pregnant women was shown in Table 1. Thirty-seven cases of pregnant women with abnormal glucose tolerance were included into the GDM group, and 36 cases of pregnant women with normal glucose tolerance (NGR) were included into NGR group. Subsequently, each group was divided into two subgroups of EBMI and NW according to pre-pregnant BMI. Seventeen cases in the GDM group were included into GDMEBMI subgroup (pre-pregnancy BMI 25 kg/m2) and 20 cases were included into GDM-NW sub-group (pre-pregnancy BMI < 25 kg/m2). The control group consisted of 20 cases in NGR-EBMI subgroup and 16 cases in NGR-NW sub-group. Comparisons of the glucolipid metabolism, HOMA-IR and serum vaspin levels between GDM and NGR groups were shown in Table 2. The HDL-C level in NGR group were significantly higher Table 1 Comparison of general condition between the two groups. Factor
N Age (year) Gravidity (median-range) Gestational age(day) BMI
NGR
GDM
NGR-NW
NGR-EBMI GDM-NW
EBMI-GDM
16 31.8 3.4 1 (1–1) 272.6 3.8 22.6 1.0
20 31.3 4.3 1 (1–1) 274.0 3.6 27.7 2.9
19 32.1 3.7 1 (1–2) 272.7 6.8 27.8 2.4
18 32.4 4.0 1 (1–1) 271.9 3.8 23.2 0.9
NGR: normal glucose tolerance; GDM: gestational diabetes mellitus; NW: normal weight; BMI: body mass index; EBMI: elevated BMI.
W.Q. Mm et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 98–101
100
than those in GDM group (P = 0.0095). While, the serum vaspin level in NGR group were significantly lower than those in GDM group (P = 0.0012). However, there were no statistical differences in TC, TG, LDL-C, FPG, FINS and HOMA-IR between these two groups (P > 0.05). A comparison was carried out within the GDM group, and result showed that there were no statistical differences in all indexes of glucolipid metabolism, HOMA-IR and serum vaspin level between subgroups (P > 0.05). However, TC, FPG, FINS and HOMA-IR in NGR-EBMI subgroup were significantly higher than those in NGR-NW subgroup (P < 0.05). Vaspin mRNA expression level in subcutaneous adipose tissue in the GDM group was significantly higher than that in the NGR group (P = 0.0003; Fig. 1). In addition, there was significant difference in the two groups (NW-NGR versus EBMI-NGR, P = 0.000; NW-GDM versus EBMI-GDM, P = 0.000). However, there was no significance in EBMI-NGR and EBMI-GDM (P = 0.086). Similarly, protein expression levels of vaspin in subcutaneous adipose tissues in the GDM group presented higher level than that in the NGR group (P = 0.025). Moreover, there were also significant differences in subgroups of GDM and NGR (NW-NGR versus EBMINGR, P = 0.016; NW-GDM versus EBMI-GDM, P = 0.035). PCA indicated that serum vaspin level was not significantly correlated with FINS and HOMA-IR in GDM women in all groups; however, there was a significant positive correlation between serum vaspin level and birth-weight (r = 0.266, P = 0.023). Comments As a recently discovered adipokine, the specific mechanism of vaspin is not yet fully determined. Current studies mostly focus on T2DM and obese patients, and the vaspin is considered as an insulin sensitizer which may be involved in regulation of endogenous glucose metabolism. Recently, more and more researchers began to study the roles of vaspin in GDM. In this study, we evaluated the serum vaspin levels and mRNA and protein expression levels of vaspin in subcutaneous adipose tissue in pregnant women with GDM or NGR. Our results suggested that vaspin levels in both serum and adipose tissue were significantly higher in GDM than those in NGR, however, serum vaspin level was not significantly correlated with FINS and HOMA-IR in GDM women, but had a positive correlation with birth-weight. Vaspin, a secretory protein, is mainly synthesized by the visceral white adipose tissue [24]. Using enzyme linked immunosorbent assay (ELISA), Western blot and RT-PCR techniques, Tan et al. [25] found that patients with polycystic ovary syndrome (PCOS) have increased serum vaspin levels and higher vaspin mRNA expression levels in omental adipose tissues. The same findings were revealed in our study. The vaspin mRNA and protein
Fig. 1. Comparison of vaspin mRNA expression levels in subcutaneous adipose tissue between the four subgroups. NW-NGR represents normal weight with normal glucose tolerance; EBMI-NGR represents elevated BMI with normal glucose tolerance; GDM represents gestational diabetes mellitus; NW-GDM represents normal weight with gestational diabetes mellitus, EBMI-GDM represents elevated BMI with gestational diabetes mellitus. BMI represents body mass index; # represents P < 0.05 compared to other three groups; * represents P < 0.001 compared to other three groups.
expressions in subcutaneous adipose tissues of GDM patients were significantly higher than those in the NGR group, and correspondingly, the serum vaspin levels in GDM patients were also significantly higher than those in the NGR group. Both GDM and PCOS patients have insulin resistance and would develop T2DM in a few years (pre-diabetic state). Moreover, PCOS women during pregnancy are also more prone to develop GDM. Therefore, we hypothesized that their pathogenesis might have some similarity. However, further studies are needed to confirm this hypothesis. In the non-pregnant state, elevated blood glucose levels are positively correlated with elevated serum vaspin levels in obese or diabetic patients [7], and serum vaspin levels are significantly decreased after metformin treatment [26]. Blood glucose and serum vaspin levels are lower, and serum insulin levels are higher in pregnant women compared with non-pregnant women [4]. Our results showed that, in late pregnancy, serum vaspin levels in GDM women were higher than those in the NGR group, but were not significantly correlated with serum FINS and HOMA-IR. This was consistent with an in vitro experimental result which demonstrated that vaspin levels were increased by adding glucoses in the adipose tissues, while not changed when adding insulin [25]. Stepan et al. found that there was no significant difference in serum vaspin level between GDM and non-GDM women, and the
Table 2 Comparison of the glucolipid metabolism and serum vaspin levels between the two groups. Factor
TC (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) FPG (mmol/l) FINS (mU/L) HOMA-IR Vaspin (ng/ml) *
NGR
GDM
NGR-NW
NGR-EBMI
GDM-NW
GDM-EBMI
P (inter-groups)
6.3 0.9 2.9 1.1 1.9 0.3 4.0 0.9 3.4 1.0 6.2 3.1 1.0 0.6 1.3 0.7
5.7 0.8* 3.2 0.9 1.7 0.3 3.7 0.7 4.2 1.3* 15. 8 12.7** 3. 5 4.7* 1.6 0.6
5.5 1.0 3.1 1.0 1.6 0.3 3.4 0.7 4.4 1.1 7.5 5.6 1.6 1.4 2.2 1.8
6.0 1.1 3.6 1.0 1.7 0.3 3.8 0.9 4.0 0.5 7.6 3.8 1.4 0.7 3.12 2.1
0.196 0.168 0.0095 0.1508 0.1047 0.0726 0.2264 0.0012
P < 0.05 for comparison within NGR or GDM group. P < 0.001 for comparison within NGR or GDM group. NGR: normal glucose tolerance; GDM: gestational diabetes mellitus; NW: normal weight; EBMI: elevated BMI; TC: total cholesterol; TG: triglyceride; HDL-C: high density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; FPG: fasting plasma glucose; FINS: fasting insulin; HOMA-IR: homeostasis model assessment of insulin resistance. **
W.Q. Mm et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 98–101
serum vaspin level had no significant correlation with insulin sensitivity in pregnant subjects [17]. However, in our study, the serum vaspin level in GDM group was significantly higher than that in non-GDM women. We hypothesized that this might result from the elimination of influence of EBMI in our study. In the study, the GDM and NGR groups were respectively divided into subgroups according to pre-pregnancy BMI, and BMI were matched between GDM-EBMI and NGR-EBMI subgroups. In addition, we know that in late pregnancy, due to the influence of factors such as fetal weight and amniotic fluid volume, there would be a relatively large error in measuring BMI and waist–hip ratio (WHR), thus the pre-pregnancy BMI were used for grouping in this study. Furthermore, it might be considered as the compensative increase of vaspin in order to ameliorate the increased IR in GDM. Besides, this study suggested that serum vaspin level had no significant correlation with glucolipid metabolism and insulin sensitivity in pregnant women, but there were significant differences in TC, FPG, FINS and HOMA-IR in NGR-EBMI and in NGR-NW subgroup. The former result was consistent with that of Stepan et al. It was, therefore, reasonable to speculate that the serum vaspin level cannot be used as an independent predictor of IR. The latter result indicated that the glucolipid metabolism was related to EBMI. However, the serum vaspin level shows a positive correlation with IR in the non-pregnant population of diabetes and obesity [7,14]. This is due to the fact that the occurrence of IR in pregnant women is very complex. In addition to adipose tissue, it has been well demonstrated that vaspin is also expressed in human and rat placenta. Besides, vaspin increases gradually during pregnancy, and elevates to the highest level at the end of gestation [27].Therefore, the placenta cannot be ignored owing to its effect of endocrine. Moreover, it has a considerable influence on glucolipid metabolism and IR during gestation period. Furthermore, we also found that there was a positive correlation between serum vaspin level and birth-weight. A previous study suggested that a positive correlation between maternal and fetal vaspin concentrations, which implied a transplacental transport of adipocytokines and speculated that vaspin may derive from fetal and/or maternal tissues [28]. In addition, other studies have recently indicated that the mRNA of vaspin level in adipose tissue is related to obesity and glucose metabolism [7,13]; therefore, we suspected that the birth-weight of infants may be indirectly influenced by maternal vaspin level. However, in this study, we did not measure vaspin mRNA and protein expression levels in placental tissues and infants, therefore it was unable to assess their roles in IR, and the regulatory mechanism (e.g. birth-weight of infants) for serum vaspin level was also not very clear. In summary, the serum vaspin levels do not reflect the degrees of IR in GDM women, that is to say, the serum vaspin level cannot serve as an independent predictor of IR. However, the serum vaspin level increases with mRNA expression level of the vaspin in subcutaneous adipose tissues in GDM women, indicating that the vaspin may be involved in the pathogenesis of GDM. In view of the relatively complex etiology of GDM, we only know that the vaspin might be involved in the occurrence of GDM, but its specific regulatory mechanism needs further study. Conflict of interest All authors declare that there is no conflict of interest in this study. Condensation Serum vaspin level is significantly higher in GDM group than those in NGR groupand is not significantly correlated with FINS and HOMA-IR.
101
References [1] Gilmartin AB, Ural SH, Repke JT. Gestational diabetes mellitus. Rev Obstet Gynecol 2008;1(3 (Summer)):129–34. [2] Butte NF. Carbohydrate and lipid metabolism in pregnancy: normal compared with gestational diabetes mellitus. Am J Clin Nutr 2000;71(5 Suppl (May)): S61–1256. [3] Briana DD, Malamitsi-Puchner A. Reviews: adipocytokines in normal and complicated pregnancies. Reprod Sci 2009;16(10 (Oct)):921–37. [4] Giomisi A, Kourtis A, Toulis KA, et al. Serum vaspin levels in normal pregnancy in comparison with non-pregnant women. Eur J Endocrinol 2011;164(4): 579–83. [5] Hida K, Wada J, Eguchi J, et al. Visceral adipose tissue-derived serine protease inhibitor: a unique insulin-sensitizing adipocytokine in obesity. Proc Natl Acad Sci USA 2005;102(30):10610–15. [6] Bluher M. Vaspin in obesity and diabetes: pathophysiological and clinical significance. Endocrine 2012;(2):176–82. [7] Youn BS, Kloting N, Kratzsch J, et al. Serum vaspin concentrations in human obesity and type 2 diabetes. Diabetes 2008;57(2):372–7. [8] Li Z, Ma C, Li L, Pan X, Chen L. Vaspin serum concentration in patients with type 2 diabetes and carotid plaque. J Int Med Res 2012;40(5):1670–6. [9] Esteghamati A, Noshad S, Mousavizadeh M, Zandieh A, Nakhjavani M. Response: association of vaspin with metabolic syndrome: the pivotal role of insulin resistance. Diabetes Metab J 2014;38(3):242–3. [10] Jia X, Wang S, Ma N, et al. Comparative analysis of vaspin in pregnant women with and without gestational diabetes mellitus and healthy non-pregnant women. Endocrine 2014;15 [Epub ahead of print]. [11] Aust G, Richter O, Rohm S, et al. Vaspin serum concentrations in patients with carotid stenosis. Atherosclerosis 2009;204(1):262–6. [12] Zvonic S, Lefevre M, Kilroy G, et al. Secretome of primary cultures of human adipose-derived stem cells: modulation of serpins by adipogenesis. Mol Cell Proteomics 2007;6(1):18–28. [13] Kloting N, Berndt J, Kralisch S, et al. Vaspin gene expression in human adipose tissue: association with obesity and type 2 diabetes. Biochem Biophys Res Commun 2006;339(1):430–6. [14] Suleymanoglu S, Tascilar E, Pirgon O, Tapan S, Meral C, Abaci A. Vaspin and its correlation with insulin sensitivity indices in obese children. Diabetes Res Clin Pract 2009;84(3):325–8. [15] Teshigawara S, Wada J, Hida K, et al. Serum vaspin concentrations are closely related to insulin resistance, and rs77060950 at SERPINA12 genetically defines distinct group with higher serum levels in Japanese population. J Clin Endocrinol Metab 2012;97(7 (Jul)):E1202–07. [16] Handisurya A, Riedl M, Vila G, et al. Serum vaspin concentrations in relation to insulin sensitivity following RYGB-induced weight loss. Obes Surg 2010;20(2 (Feb)): 198–203. [17] Stepan H, Kralisch S, Klostermann K, et al. Preliminary report: circulating levels of the adipokine vaspin in gestational diabetes mellitus and preeclampsia. Metabolism 2010;59(7):1054–6. [18] Gkiomisi A, Makedou KG, Anastasilakis AD, et al. Serum vaspin levels in women with and without gestational diabetes mellitus during pregnancy and postpartum. Cytokine 2013;61(1 (Jan)):127–32. [19] Metzger BE, Gabbe SG, Persson B, et al. International association of diabetes and pregnancy study groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 2010;33(3): 676–82. [20] Metzger BE, Coustan DR. Summary and recommendations of the Fourth International Workshop-Conference on gestational diabetes mellitus. The Organizing Committee. Diabetes Care 1998;21(Suppl 2 (Aug)): B161–7. [21] Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. National Diabetes Data Group. Diabetes 1979;28(12): 1039–57. [22] Carpenter MW, Coustan DR. Criteria for screening tests for gestational diabetes. Am J Obstet Gynecol 1982;144(7):768–73. [23] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28(7):412–9. [24] Hida K, Wada J, Zhang H, et al. Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats. J Lipid Res 2000;41(10): 1615–22. [25] Tan BK, Heutling D, Chen J, et al. Metformin decreases the adipokine vaspin in overweight women with polycystic ovary syndrome concomitant with improvement in insulin sensitivity and a decrease in insulin resistance. Diabetes 2008;57(6):1501–7. [26] Gulcelik NE, Karakaya J, Gedik A, Usman A, Gurlek A. Serum vaspin levels in type 2 diabetic women in relation to microvascular complications. Eur J Endocrinol 2009;160(1):65–70. [27] Caminos JE, Bravo SB, Garces MF, et al. Vaspin and amylin are expressed in human and rat placenta and regulated by nutritional status. Histol Histopathol 2009;24(8):979–90. [28] Briana DD, Boutsikou M, Baka S, et al. Omentin-1 and vaspin are present in the fetus and neonate, and perinatal concentrations are similar in normal and growth-restricted pregnancies. Metab Clin Exp 2011;60(4): 486–90.
