The Role of Adipocytokines in Insulin Resistance in Normal Pregnancy: Visfatin Concentrations in Early Pregnancy Predict Insulin Sensitivity

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1 Papers in Press. Published June 22, 2007 as doi: /clinchem The latest version is at Clinical Chemistry 53: (2007) Endocrinology and Metabolism The Role of Adipocytokines in Insulin Resistance in Normal Pregnancy: Visfatin Concentrations in Early Pregnancy Predict Insulin Sensitivity George Mastorakos, 1 Georgios Valsamakis, 1 Dimitrios Papatheodorou, 2 Ioannis Barlas, 1 Alexandra Margeli, 3 Anastasios Boutsiadis, 1 Evangelia Kouskouni, 1 Nikolaos Vitoratos, 1 Apostolos Papadimitriou, 2 Ioannis Papassotiriou, 3* and George Creatsas 1 Background: Throughout pregnancy maternal adipose tissue is metabolically active, producing adipocytokines involved in the process of insulin resistance. We explored the role of serum adipocytokines, including the newly identified adipocytokine visfatin, in the process of insulin resistance in normal pregnancy. Methods: We examined 80 pregnant nonobese, nondiabetic white women during the 3 trimesters of pregnancy. All study participants underwent anthropometric measurements, adipocytokine evaluation, and a 75-g oral glucose tolerance test. Homeostasis mathematical model assessment (HOMA-R), insulin sensitivity index (ISI), and indices of -cell secretion were calculated. Results: Maternal weight, percentage total body fat, hip circumference, and indices of -cell secretion increased significantly during the 3 trimesters, and HOMA-R and ISI increased and decreased, respectively, in the 3rd trimester. During early pregnancy, insulin resistance, -cell secretion, and weight correlated positively with leptin. During the 1st trimester, visfatin correlated negatively with percentage 1 Endocrine Unit, 2nd Department of Obstetrics and Gynecology, Aretaieion University Hospital, Athens Medical School, Athens, Greece. 2 Department of Obstetrics and Gynecology, Nikaia General Hospital, Nikaia, Greece. 3 Department of Clinical Biochemistry, Aghia Sophia Children s Hospital, Athens, Greece. * Address correspondence to this author at: Department of Clinical Biochemistry, Aghia Sophia Children s Hospital, Athens, Greece. Fax ; biochem@paidon-agiasofia.gr or jpapasotiriou@ath. forthnet.gr. Received December 17, 2006; accepted May 22, Previously published online at DOI: /clinchem body fat and was the best positive predictor of 2nd trimester ISI. In the 2nd trimester, serum visfatin was the best negative predictor of percentage body fat. Conclusions: During normal pregnancy of nonobese, nondiabetic women, adipose tissue increases, accompanied by a significant progressive increase of insulin resistance. Visfatin concentrations in the 1st trimester positively predict insulin sensitivity during the 2nd trimester. Body fat mass during 1st trimester of pregnancy is negatively associated with insulin sensitivity during the 2nd trimester and perhaps should be kept under control American Association for Clinical Chemistry During pregnancy, changes in maternal metabolism occur in response to the growing fetus and placenta and their increasing metabolic needs. Pregnancy is associated with alterations in the regulation of glucose metabolism caused by the actions of human placental growth hormone, prolactin, cortisol, and progesterone; these hormones antagonize the action of insulin, particularly during the 2nd and 3rd trimesters, leading to a state of relative insulin resistance as pregnancy progresses (1). The mother s body adapts to the new metabolic environment so that the transfer of glucose to the fetus is adequate. Redistribution of maternal adipose tissue occurs throughout pregnancy (2). The adipose tissue is not just a storage depot but is also a metabolically active tissue, producing adipocytokines that exert endocrine and paracrine effects (3). Adipocytokines such as leptin and adiponectin are involved in the process of insulin resistance and energy homeostasis. Leptin concentrations are increased throughout pregnancies associated with pre- 1 Copyright (C) 2007 by The American Association for Clinical Chemistry

2 2 Mastorakos et al.: Visfatin in Normal Pregnancy eclampsia, body mass index (BMI) 4 25 kg/m 2, gestational diabetes, or hyperinsulinemia (4 7). Decreased serum adiponectin concentrations characterize women with a history of gestational diabetes independently of insulin sensitivity or the degree of obesity. In fact these concentration changes are associated with subclinical inflammation (8). In late pregnancy the human placenta produces and secretes adiponectin, and adiponectin and its receptors are differentially regulated by cytokines (9). Furthermore, inflammatory molecules such as interleukin-6 (IL-6) and C-reactive protein (CRP) are involved in the process of insulin resistance and are found to be increased in maternal serum and peritoneal washing fluid during arrested labor (10). A new adipocytokine, visfatin, which may have insulinomimetic action, was recently isolated. Visfatin is expressed in the visceral fat of humans, and its plasma concentrations increase during the development of obesity (11). The release of visfatin may be regulated by glucose and insulin (12), and it increases with progressive -cell deterioration (13). Furthermore, visfatin may possess proinflammatory and immunomodulating properties (14). In a study of Asian Indian immigrants, visfatin was shown to be related to HDL metabolism (15). There are reports of increased visfatin plasma concentrations in gestational (16), type 1 (13) or type 2 (17) diabetes, and obesity (18). Other studies, however, have revealed opposite effects in gestational diabetes (19) and obesity (20). The aim of this study was to explore the effect of adipose tissue in the development of insulin resistance in healthy nonobese and nondiabetic pregnant women. 4 Nonstandard abbreviations: BMI, body mass index; IL, interleukin; CRP, C-reactive protein; hs, high-sensitivity; OGTT, oral glucose tolerance test; HOMA-R, homeostasis mathematical model assessment; ISI, insulin sensitivity index; PHIS, phase of insulin secretion; AUCI, of the area under the curve of insulin. Materials and Methods patient recruitment The study had the approval of the local ethics committee, and all study participants gave written informed consent. Study participants were 80 pregnant primigravidae white women with mean (SD) BMI of 24.7 (2.2) kg/m 2 (before pregnancy) and age of 29.5 (4.5) years, with no history of type 2 diabetes mellitus, recruited during the 1st trimester of pregnancy from the Obstetrics and Gynecology outpatient clinic of Aretaieion University Hospital (Athens) and the Nikaia General Hospital (Athens). Exclusion criteria were as follows: nonwhite; BMI before pregnancy 30 kg/m 2 ; history of diabetes type 1 or type 2; multiple pregnancy; major vaginal bleeding; hypertension; preeclampsia; urinary tract infection; fever ( 37.5 C); fetal/placental abnormalities (i.e., congenital anomalies, placenta previae, placental abruption, intrauterine retardation); remarkable previous medical, surgical, and gynecological maternal history; and current smoking or alcohol intake. To avoid bias, women were recruited on a random, consecutive 1st-visit basis. protocol The recruited pregnant women were seen in the outpatient clinic once during each of the 3 trimesters of their pregnancy at weeks 10 12, 24 26, and 34 36, respectively. Five of the women who were selected for study participation developed gestational diabetes during pregnancy and were subsequently excluded. At each visit, study participants underwent the following clinical and biochemical examinations. anthropometric measurements All measurements were performed by a single observer. Weight in kilograms to the nearest 0.1 kg on a beam balance was measured for study participant without shoes and dressed in light clothing. Height in meters was measured to the nearest millimeter with a stadiometer, and the BMI in kilograms per square meter was calculated. Maximum hip circumference in centimeters was measured in duplicate with a 6-mm-wide flexible tape at the widest part of the trochanters while the study participant was in a horizontal position with feet kept cm apart. Skinfold thicknesses were measured on the left side of the body with a Harpenden skinfold caliper (Assist Creative Resources Ltd.) in triplicate to the nearest 0.2 mm. Biceps and triceps thicknesses were measured at the midpoint of the upper arm, between the acromion process and the tip of the bent elbow. Subscapular skinfold thickness was measured at the natural fold 2 3 mm below the shoulder blade at an oblique angle. The suprailiac skinfold was pinched at 2 3 cm above the iliac crest on the lateral side and midaxillary line. The sum of skinfold measurements made at all 4 locations was estimated and used to determine percentage body fat with charts interpolating for age based on data from Durnin and Womersley (21). Supine blood pressure was recorded as the mean of 3 measurements made with a mercury sphygmomanometer. biochemical assays After study participants had fasted overnight, they underwent a 75-g oral glucose tolerance test (OGTT) and blood samples were drawn at 9:00 AM for measurement of glucose, lipids, insulin, IL-6, leptin, adiponectin, highsensitivity (hs)crp, and visfatin at the time points of 0 min and at 5, 15, 30, 60, 90, and 120 min for measurement of insulin and glucose. Blood samples were stored at 70 C. Insulin was measured with the Medgenic immunoenzymetric assay (Biosource-Europe SA) and hscrp with a highly sensitive latex-particle-enhanced immunonephelometric assay on the BN ProSpec nephelometer (Dade Behring). We measured serum leptin with an RIA (Linco Research) and serum IL-6 with the Quantikine hs human IL-6 ELISA (R&D Systems; according to the man-

3 Clinical Chemistry 53, No. 8, ufacturer, the limit of quantification is ng/l and the inter- and intraassay CVs at ng/l are 9.6% and 6.9%, respectively). Serum adiponectin was determined using an ELISA from Chemicon International. For the adiponectin assay, according to the manufacturer, the limit of quantification of the assay was 0.1 g/l with a range of calibrators from 0.23 to 15 g/l, and inter- and intraassay CVs were 9.8% (7.5 g/l) and 8.4% (3.7 g/l), respectively (22). Serum visfatin concentrations were measured with an RIA from Phoenix Pharmaceuticals (inter- and intraassay CVs, according to the manufacturer, were 6%). Aprotinin was added to all samples. indices of carbohydrate metabolism Carbohydrate metabolism index derived from fasting values. Insulin resistance was derived from fasting glucose and insulin concentrations by use of the homeostasis mathematical model assessment (HOMA-R) [insulin at baseline (pmol/l) glucose at baseline (mmol/l)]/135 (23). Carbohydrate metabolism indices derived from OGTT results. Insulin sensitivity was estimated by use of the insulin sensitivity index (ISI) [ BMI] [ insulin at 120 min (pmol/l)] [ glucose at 90 min (mmol/l)] (24). -Cell secretion of insulin was estimated by the following indices (24): predicted index of the 1st phase of insulin secretion (1st PHIS) 1283 [1.289 insulin at 30 min (pmol/l)] [138.7 glucose at 30 min (mmol/l)] [3.772 insulin at baseline (pmol/l)] and predicted index of the 2nd PHIS 287 [ insulin at 30 min (pmol/l)] [26.07 glucose at 30 min (mmol/l)] [ insulin at baseline (pmol/l)]. Hyperinsulinemia was estimated by the of the area under the curve of insulin ( AUCI). The AUCI during OGTT was calculated after subtraction of the insulin value at 0 min from each of the 5, 15, 30, 60, 90 and 120 min insulin values by application of the trapezoidal rule of the area of calculation. statistical analysis Data are described as mean (SD) or as median and interquartile range for data with nongaussian distribution. To test the change of each variable during pregnancy, the one-way repeated measures ANOVA test was used for variables with gaussian distribution and the nonparametric Friedman ANOVA test for those with nongaussian distributions. To test the associations between variables, we used Spearman correlation analysis. Backwards regression analysis was used to define predictive variables. Longitudinal (univariate, bivariate, and multiple) models using fixed effects were performed with time taken into consideration. Random effects were examined and were not found to be statistically significant. Therefore, these effects were not included in the models. Time was found to be a significant variable in univariate models and was subsequently included in all bivariate models. A P value of 0.05 was considered significant. P values were automatically calculated when correlation coefficients or regression analyses were performed with the SPSS statistical package that was used for statistical analyses (SPSS) (25). Table 1. Anthropometric and metabolic variables during pregnancy. a n 80 1st trimester 2nd trimester 3rd trimester Weight, kg 67.6 (13.4) 76 (12.5) * 84.4 (15.6) * % Fat 30.4 (5.2) 32.2 (4.9) * 34.4 (6.9) * Hip circumference, cm (12.1) (10.2) * 116 (11.8) * Skinfold biceps, mm 16.5 ( ) 20 ( ) * 25.6 (16 29) * Skinfold triceps, mm 16.2 ( ) 21.5 (17 25) * 23 (16 29) * Skinfold suprailiac, mm 12.2 (8.5 13) 13.5 ( ) * 16 ( ) * Skinfold subscapular, mm 16.7 ( ) 18 (16 27) 22.2 (14 31) * Visfatin, g/l 23.7 ( ) 26 ( ) * 27.1 ( ) * Adiponectin, mg/l 5.2 ( ) 5.1 ( ) 6.3 ( ) Leptin, g/l 25.9 ( ) 44.5 ( ) 53 ( ) IL-6, ng/l 1.8 ( ) 2 ( ) 1.7 ( ) hscrp, mg/l 2.3 ( ) 2.1 ( ) 2.6 ( ) Glucose 0 min, mmol/l 4.5 (0.4) 4.4 (0.4) 4.4 (0.4) Insulin 0 min, pmol/l 58.4 ( ) 58.8 ( ) 115 (86 145) * ISI 0.1 ( ) 0.08 ( ) 0.05 ( ) * HOMA-R 1.9 ( ) 1.8 ( ) 3.7 ( ) * PHIS ( ) 2755 ( ) * 4166 ( ) * PHIS2 696 ( ) 764 ( ) * 1087 ( ) * AUCI 9884 ( ) ( ) ( ) a Variables expressed as mean (SD) or median (25th 75th interquartile range). *, Statistically significant change from 1st trimester., Statistically significant change from 2nd trimester.

4 4 Mastorakos et al.: Visfatin in Normal Pregnancy as compared with 1st and 2nd trimesters, whereas the ISI decreased significantly in 3rd as compared with 1st and 2nd trimesters (P 0.02; Table 1). -Cell secretion indices 1st PHIS (P ) and 2nd PHIS (P 0.03) increased gradually but significantly from the 1st to the 2nd and 3rd trimester of pregnancy, indicating a sustained -cell function (Table 1). There was a nonsignificant upward trend in AUCI through the 3 trimesters (Table 1). Fig. 1. Change of serum visfatin:isi ratio during normal pregnancy. The asterisk indicates statistically significant difference (P 0.05) from the 1st and 2nd trimester mean values. Results changes of anthropometric variables and adipocytokines during pregnancy Maternal weight (P 0.005), percentage total body fat (P 0.042), and hip circumference (P 0.039) increased gradually but significantly from the 1st to the 2nd and 3rd trimesters of pregnancy (Table 1). Body fat mass indicated by hip circumference and in the suprailiac area and biceps increased significantly during each trimester from the 1st to the 3rd. Triceps and subscapular skinfolds increased only from the 1st to the 2nd trimester (Table 1). Mean visfatin concentrations were significantly higher during the 2nd and 3rd trimesters of pregnancy compared with the 1st trimester (P 0.041; Table 1). Interestingly, the mean ratios of visfatin to ISI were significantly higher during the 3rd trimester than the 1st (P 0.015) and 2nd trimesters (P 0.019; Fig. 1). Maternal adiponectin, leptin, IL-6, and hscrp did not change significantly during pregnancy, but there was a clear, nonsignificant upward trend in mean leptin concentrations (Table 1). changes of carbohydrate metabolism parameters during pregnancy Fasting insulin concentrations at 0 min (P 0.03) and HOMA-R index (P 0.03) were significantly higher in 3rd correlations among carbohydrate metabolism parameters, adipocytokines, and anthropometric variables during the 3 trimesters of pregnancy Significant correlations (P 0.05) among carbohydrate metabolism indices and anthropometric variables through the 3 trimesters of pregnancy are presented in Table 2. Longitudinal univariate models showed a significant negative change of ISI with time as an independent variable (P between 2nd and 1st trimesters; P between 3rd and 1st trimesters), with percentage fat as an independent variable (P 0.003), and with hip circumference as an independent variable (P 0.001). Longitudinal bivariate models showed a significant negative change of ISI with time and percentage fat taken together as independent variables (P 0.015), and with time and hip circumference taken together as independent variables (P 0.004). Interestingly, most of the correlations among these parameters were noted during the 1st and 2nd trimesters. Significant correlations (P 0.05) among carbohydrate metabolism indices and adipocytokines throughout the 3 trimesters of pregnancy are presented in Table 3. IL-6 showed no statistically significant correlation with the variables examined (P 0.05). Significant correlations (P 0.05) among anthropometric variables and adipocytokines through the 3 trimesters of pregnancy are presented in Table 4. Of note, serum leptin correlated positively with weight before pregnancy and at the 1st trimester, confirming the known positive association between this adipocytokine and body fat mass. Finally, leptin concentrations during the 1st and 2nd trimesters correlated positively with hscrp (r 0.59, P 0.009, and r 0.50, P 0.017, respectively). Table 2. Statistically significant (P <0.05) correlations among carbohydrate metabolism and anthropometric variables. a 1st trimester Variable 1 ISI ISI ISI 1st PHIS 1st PHIS 1st PHIS 2nd PHIS 2nd PHIS 2nd PHIS Variable 2 Weight before Weight Hip Weight before Weight % Body fat Weight before Weight % Body fat r-spearman nd trimester 3rd trimester Variable 1 ISI 1st PHIS 1st PHIS 2nd PHIS ISI ISI Variable 2 Weight Weight % Body fat Weight % Body fat Hip r-spearman a The correlation is expressed by the r-spearman, which becomes meaningful at P 0.05.

5 Clinical Chemistry 53, No. 8, Table 3. Statistically significant correlations among carbohydrate metabolism variables and adipocytokines. a 1st trimester Variable 1 1st PHIS 1st PHIS 2nd PHIS 2nd PHIS AUCI AUCI ISI ISI Variable 2 Leptin HsCRP Leptin hscrp Leptin hscrp Leptin hscrp r-spearman nd trimester Variable 1 1st PHIS 2nd PHIS AUCI Variable 2 Leptin Leptin Leptin r-spearman a The correlation is expressed by the r-spearman is significant at P predictors of insulin sensitivity and percentage body fat during the 2nd trimester Backward multiple regression analysis models revealed that of the 1st trimester variables hscrp, adiponectin, IL-6, leptin, weight before pregnancy, body fat percentage, and vistafin, serum visfatin concentration during the 1st trimester ( 1.018, P 0.01) was the best positive predictor of the 2nd trimester ISI (Table 5) and of the 2nd trimester variables adiponectin, leptin, hscrp, IL-6, and vistafin, fasting serum visfatin ( 1.1, P 0.016) was the best negative predictor of percentage body fat. Of note, longitudinal models have shown that visfatin was not significantly correlated with ISI over all 3 time-points (trimesters) examined, whereas hip circumference (P 0.005) and percentage body fat (P 0.015), separately examined, have shown a significant negative correlation with ISI when time and visfatin were taken into consideration. Discussion To investigate the effect of adipose tissue on insulin resistance in normal pregnancy during all 3 trimesters in a single group of nonobese, nondiabetic healthy women, we used mathematically derived OGTT-based indices (23, 24) that have been validated in previous studies (26 28). We observed a significant progressive increase of insulin resistance and decrease of insulin sensitivity from the 1st to the 3rd trimester. Recently, Endo et al. (29) showed in 3 different groups of pregnant normal-weight nondiabetic women, 1 group studied for each of the 3 trimesters of pregnancy, that HOMA-R was unchanged throughout gestation, whereas -cell secretion increased. The discrepancy between their results and ours may be attributable to their use of a group of women for each trimester. Furthermore, we found that estimated fat percentage increased significantly during pregnancy. This estimation is only an approximation of body fat mass but is the only ethical method for measurement of body fat mass in pregnant women. Sidebottom et al. (30) found that subcutaneous maternal body fat stores remain stable during the 1st 6 weeks after conception and then increase from 6 to 35 weeks, according to measurements at the triceps and particularly at the subscapular and thigh areas. Maternal serum concentrations of visfatin increased during pregnancy, especially between the 1st and 2nd trimesters, in parallel with -cell secretion indices. During the 1st trimester, visfatin correlated negatively with percentage fat mass and hip circumference, whereas during the 2nd and 3rd trimesters this negative correlation disappeared, indicating a differential quantitative change between visfatin concentrations and adipose tissue. The gradually increasing insulin resistance during pregnancy may be compensated for by a sustained increase of visfatin, an insulinomimetic molecule (11, 31). Indeed, visfatin concentration during the 1st and 2nd trimester was a predictor of 2nd-trimester ISI (positive association) and percentage total body fat (negative association), respectively. According to longitudinal multiple models analysis, visfatin and ISI did not change in a similar way during all 3 trimesters, whereas adipose tissue and ISI conserved their negative association. The loss of the close association of visfatin with ISI after the 2nd trimester may be attributable to an increase of visfatin production by an additional source other than adipose tissue, namely the placenta (Fig. 1). Interestingly, Krzyzanowska et al. (16) have recently shown that serum visfatin concentrations increase significantly during the course of pregnancies in women who developed gestational diabetes. Alternatively, Berndt et al. (18) recently reported that in nonpreg- Table 4. Statistically significant correlations among anthropometric parameters and adipocytokines. a 1st trimester Variable 1 Weight Weight Weight % Hip before before Body fat Variable 2 Leptin Visfatin Leptin Visfatin Visfatin r-spearman a The r-spearman correlation is significant at P Table 5. Backwards multiple regression analysis for dependent variable ISI 2nd trimester. a Coefficient SE of coefficient P value Visfatin 1st trimester HsCRP 1st trimester a The term coefficient is the generally used term for the effect of each predictor (independent variable) on the respective dependent variable in a linear regression analysis.

6 6 Mastorakos et al.: Visfatin in Normal Pregnancy nant women with a wide range of obesity, visfatin plasma concentrations did not correlate with percentage body fat or waist-to-hip ratio. Leptin concentrations reflected hyperinsulinemia ( AUCI) and insulin resistance (ISI) during the 1st and 2nd trimesters, confirming previous studies conducted in healthy pregnant women, with separate study groups for each trimester (32), as well as in pregnant women with type 1 and type 2 diabetes and gestational diabetes mellitus (4 7). The clear upward trend in leptin concentrations throughout the 3 trimesters, although not statistically significant, parallels the increase of percentage fat mass. In this study, serum adiponectin concentrations did not change significantly during pregnancy, whereas other studies of separate groups of women for each trimester have shown a decrease of adiponectin concentrations in late normal pregnancy by (33, 34). The lack of a decrease of adiponectin concentrations throughout pregnancy in our study population, in contrast to the known decrease of this adipocytokine in pregnant women with type 1, type 2, or gestational diabetes, is probably attributable to the fact that the women participating in our study were healthy, with normal BMI. Alternatively, the absence of decrease of adiponectin throughout normal pregnancy may represent an independent protective mechanism against the gradual increase of insulin resistance. Furthermore, in this study adiponectin concentrations did not correlate with markers of insulin sensitivity. In studies of normal pregnancies, discordant results have been observed regarding the association of adiponectin concentrations with ISI (26), whereas adiponectin is increased in preeclampsia (35), a state associated with hyperinsulinemia (36, 37). Insulin resistance in normal pregnancy seems to increase throughout the 2nd and 3rd trimesters, and body fat mass increases from the 1st trimester throughout pregnancy, indicating that the latter might be a causal factor for the decrease of insulin sensitivity. Thus, close monitoring of maternal body fat mass as early as the 1st trimester may be warranted even during normal uncomplicated pregnancies of nonobese women. It would be of interest to compare our findings with those for pregnancies in nondiabetic obese women and in women with gestational diabetes. Insulin secretion and resistance indices correlate with anthropometry mainly in early pregnancy, and these correlations do not persist during the 2nd and 3rd trimesters, probably because of the full formation of placenta and the independent effect of placental hormones on carbohydrate metabolism (38). Identification of predictors of insulin sensitivity associated with adipose tissue, such as the adipocytokine visfatin, that occur early in pregnancy may elucidate the pathogenesis of insulin resistance, leading to the discovery of methods to prevent diabetic pregnancy complications. In conclusion, during normal pregnancy in nonobese women, increased adipose tissue is a forerunner of significant progressive increase of insulin resistance. The 1st trimester concentrations of adipose tissue-derived visfatin, an insulinomimetic adipocytokine, predict insulin sensitivity during the 2nd trimester. Visfatin concentration is not yet useful as a diagnostic tool, however, because of large variations in visfatin data that require further investigation in studies with larger numbers of participants and more accurate assays. Grant/funding support: Funding was received from Hellenic Endocrine Society (to G.M.). 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