SERUM ADROPIN AND ADIPOSE FATTY-ACID BINDING PROTEIN AT 6 TH WEEK OF PREGNANCY ARE SIGNIFICANT PREDICTORS FOR DEVELOPMENT OF INSULIN RESISTANCE AT THE 24 TH WEEK

Amr Sharafeldeen MD 1 and Raafat R. Mohammed MD 2 . 1. Departments of Obstetrics & Gynecology, Faculty of Medicine, Benha University. 2. Fellow & Researcher of Medical Biochemistry, Hospital Lab, Clinical Pathology department, Faculty of Medicine, Benha University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Fatty-acid-binding proteins (FABP) are small intracellular proteins of 14-15 KDa expressed in several tissues (6) .Adipocyte FABP (AFABP) is mainly expressed in adipocytes and macrophages where it regulates adipocyte fattyacid uptake, lipogenesis and delivery of lipids to nuclear receptors to mediate nuclear transcriptional programs (7) .In macrophages AFABP modulates inflammatory responses and cholesterol ester accumulation (8) .Despite being intracellular proteins, FABP are released into the circulation and its increased plasma levels had found in several clinical conditions and proposed as markers of tissue injury (6) .Plasma AFABP levels are increased in metabolic disorders as obesity, type-2 diabetes mellitus (9) and cardiovascular conditions (10) , and in critically-ill patients and correlate with poor prognosis (11) .
New peptides not secreted by adipose tissue have a significant role in metabolic regulations (3) .Adropin (ADR) is a secreted peptide translated from the Energy Homeostasis Associated (ENHO) gene (12) which comprised two-exons on human chromosome 9p13.3 (13)and linked to metabolic control and vascular function (14) .ADR is a 42-aminoacid peptide hormone which is highly conserved across mammalian species through open reading frame in exon 2 that encodes the full-length 75-amino acid peptide; 33-amino acid as secretory signal peptide and the biologically active ADR (13) .ADR is abundant in liver and secreted into the circulation and its plasma concentrations are highly regulated by energy intake (15) and functions to preserve the circulatory system through regulating endothelial function and activity of endothelial nitric oxide (16) .

Hypothesis:-
The current study suggests a certain relation between development of gestational IR and DM on one-side and disturbed levels of plasma peptides with an action related to energy homeostasis on the other side.

Aim of work:-
This study aimed to estimate serum ADR and AFABP levels and its relations with IR as judged by the score of homeostasis model assessment of IR (HOMA-IR) in pregnant women.The study also targets to evaluate the predictive value of estimated serum ADR and AFABP levels early in pregnancy for later development of GDM.

Patients & Methods:-
After approval of the study protocol by the Local Ethical Committee, all pregnant women who attended the Antenatal Outpatient Clinics (OPC), at Benha University Hospitals since June 2015 for assurance of diagnosis of being pregnant and signed written fully informed consent to participate in the study were evaluated for eligibility for study inclusion.All women underwent evaluation for demographic data including age, weight and height, and for baseline clinical and obstetric data.Body mass index (BMI) was calculated in kg/m 2 as weight (kg)/ height (m 2 ) (17) .
Exclusion criteria include manifest DM, previous GDM in multipara women, morbid obesity with BMI>35 kg/m 2 (18) and liver, or renal diseases.All pregnant women gave blood samples for estimation of random blood glucose to assure absence of manifest DM and women were asked to attend the OPC overnight fasting on the next day to give blood samples for estimation of fasting blood glucose (FBG), and fasting serum insulin (FSI), ADR and AFABP.Then, all women underwent the 75-Oral glucose tolerance test (OGTT) which consists of estimation of FBG and postprandial blood glucose (PPBG) levels at one and two hours after taking a 75-gm oral glucose diet.All women were asked to attend the OPC overnight fasting during the period between 24 th and 28 th week GA to give blood samples for re-estimation of FSI and to repeat the 75-OGTT.
Insulin resistance (IR) was evaluated using the homeostasis model assessment of IR (HOMA-IR) score calculated according to the formula: fasting serum insulin (µU/ml) x [fasting plasma glucose (mg/ml)/18])/22.5;HOMA-IR score of >2 is considered abnormal (19) .HOMA-IR score was determined twice at 6 th and 24 th week GA.The results of the 75-OGTT were interpreted for diagnosis of GDM according to the recommendations of the International association of diabetes and pregnancy study groups (20) as follows: FBG ≥92 mg/dl, 1-h BG ≥180 mg/dl and 2-h BG ≥153 mg/dl.Investigations:-Sampling:-Venous blood samples (5 ml) were collected from the antecubital vein under complete aseptic conditions and were divided into two parts: 1.The first part was put in a tube containing sodium fluoride (2 mg sodium fluoride/ ml blood) to prevent glycolysis for estimation of blood glucose levels.
2. The second part was collected in plain tube, allowed to clot, centrifuged at 1500×g for 15 min and the serum samples were collected in clean dry Eppindorff tube to be stored at -70°C until assayed.

Statistical analysis:-
Obtained data were presented as mean±SD, numbers and percentages.Results were analyzed using One-way ANOVA with post-hoc Tukey HSD Test and Chi-square test (X 2 test).Possible relationships were investigated using Pearson's linear regression.Sensitivity & specificity of estimated parameters as predictors were evaluated using the receiver operating characteristic (ROC) curve analysis judged by the area under the curve (AUC) compared versus the null hypothesis that AUC=0.05.Statistical analysis was conducted using the IBM SPSS (Version 23, 2015) for Windows statistical package.P value <0.05 was considered statistically significant.

Results:-
The study included 348 women eligible for evaluation, 13 women were excluded and 335 women were included in the study (Fig. 1).At the 24 th wk GA, OGTT defined 39 GDM women (GDM group) for a frequency of GDM of 11.6%, while 296 women (88.4%) achieved the 24 th wk free of GDM (Non-GDM group).Baseline body weight (BW) and BMI were significantly higher in GDM than non-GDM women; otherwise there was non-significant (p>0.05)difference between studied women as regards baseline demographic and clinical data (Table 1) Mean FBG and PPBG levels estimated at the 6 th wk GA were non-significantly (p>0.05)higher in GDM women compared to non-GDM women.At the 24 th wk GA, FBG and PPBG levels were significantly higher in GDM women compared to their corresponding measures at the 6 th wk GA and to corresponding measures of non-GDM women at 24 th wk GA.On the other hand, FBG levels at the 24 th wk GA were non-significantly (p>0.05)higher, while PPBG measures were significantly higher in non-GDM women compared to their 6 th wk GA levels.At the 24 th wk GA, mean FSI levels were significantly (p<0.05)higher in all women compared to their 6 th wk GA levels with significantly higher (p<0.05)levels in GDM women than non-GDM women.Calculated HOMA-IR score defined 77 and 106 IR women, at the 6 th and 24 th wk GA, respectively with significantly higher frequency among GDM than non-GDM women, (p=0.001,respectively).Mean HOMA-IR score was significantly higher (p<0.05) in all women at the 24 th wk GA compared to their respective 6 th wk GA score with significantly higher score in GDM than non-GDM women (Table 2).Moreover, 6 th wk GA serum ADR levels were significantly (p<0.05)lower (Fig. 2), while serum AFABP levels were significantly (p<0.05)higher in GDM than non-GDM women.6 th wk GA demographic and laboratory data defined serum ADR and AFABP levels as the persistently positive predictors for development of GDM at the 24 th wk GA (Table 4).

Discussion:-
The body responses to metabolic demands of pregnancy still a challenging concern for physicians (25) and became a stressful target with the global increasing prevalence of obesity (26) .The current study assured pregnancy imposed glucogenic stresses on maternal body as evidenced by the reported high 24 th wk blood glucose levels in all the study population in relation to their baseline 6 th wk levels.Moreover, at 24 th wk, 39 women (11.6%) had abnormal OGTT and considered as having GDM.Diagnosis of GDM relied on the results of OGTT that was interpreted according to the recommendations of the International association of diabetes and pregnancy study groups (20) ; similarly multiple studies used this diagnostic policy for GDM (27,28,29) .The reported figure for frequency of GDM goes in hand with Ma et al. (30) , Karcaaltincaba et al. (31) and Arbib et al. (32) who reported frequencies of 12.2%, 11.1% and 9.9%, respectively for GDM.Moreover, Huhn et al. (33) and Brown & Wyckoff (34) documented that the introduction of the IADPSG criteria resulted in an absolute increase of GDM prevalence of 8.5% (33) and a 1.03-3.78-foldrise versus baseline criteria (34) .
As another evidence for the metabolic burden of pregnancy, IR defined by HOMA-IR score of >2 was detected in 77 and 106 women at the 6 th and 24 th wk, respectively, with an increase by 37.7%, irrespective of the development of GDM.Moreover, frequency of IR among GDM women was significantly higher than among non-GDM women.
Development of IR and DGM showed positive significant correlation with baseline maternal BMI and development of GDM showed positive significant correlation with maternal age.
These findings go in hand with Shepherd et al. (35) who detected reduced risk of GDM with their proposed diet and exercise interventions for pregnant women versus women received no intervention.Also, Pan et al. (36) reported that women with GDM were older and had higher BMI than women free of GDM and Li et al. (37) found the levels of FBG, PPBGG, FSI, lipid profile and HOMA-IR score were significantly higher in GDM than in normal glucose tolerance women with a positive correlation between HOMA-IR and BMI and concluded that with the increases of FBG, the progression of IR is increased and pancreatic β-cell function progressively declines.Recently, Lindsay et al. (38) reported a high rate of late IR among pregnant women of moderately older age and high rate of obesity.
The 6 th wk serum levels of ADR and AFABP levels were significantly lower and higher, respectively in GDM than non-GDM women and showed significant correlation with GDM, maternal age and BMI.ROC curve analysis defined high 6 th wk serum AFABP and insulin and low serum ADR levels as significant predictors for IR development at 24 th wk GA, however, Regression analysis defined high 6 th wk serum insulin as positive and low ADR levels as negative significant predictors for IR development at the 24 th wk GA.
These findings coincided with Fasshauer et al. (39) who documented that adipokines, adiponectin, leptin and AFABP seem to be the most probable candidates involved in GDM pathogenesis.Also, Zhang et al. (40) detected higher level of AFABP during mid-and late stages of pregnancy in GDM women and concluded that AFABP might be closely related to obesity and IR in pregnancy, and is a major risk factor for GDM.Thereafter, Li et al. (41) detected significantly higher expression of AFABP in serum, placenta and decidua of pregnant GDM women than in normal pregnant women.
The relation between AFABP and development of IR and GDM could be attributed to the findings provided by Garin-Shkolnik et al. (42) who proposed that suppression of peroxisome proliferator-activated receptor γ by FABP4 in visceral fat may explain the role of FABP4 in development of obesity-related morbidities, including IR.Thereafter, Li et al. (41) found serum of GDM women induced significantly increased expression of FABP4 mRNA in human pre-adipocytes and Svensson et al. (43) reported change of adipose tissue morphology and function during pregnancy, irrespective of glycemic status and supposed that increased fat mass and the proportion of very large adipocytes may contribute significantly to gestational IR and were strongly associated with late high HOMA-IR score.
In line with findings concerning serum ADR, Aydin et al. (44) , Celik et al. (45) and Beigi et al. (46) detected lower ADR levels in GDM women with significant difference versus control women.Moreover, Celik et al. (45) detected lower ADR in cord blood independent on the maternal levels and Beigi et al. (46) defined a significant association between ADR levels and GDM.
On contrary to the obtained results, Dąbrowski et al. (47) reported significantly higher ADR concentrations in GDM patients than in control group.However, against these data Gao et al. (48) tried ADR treatment of diet-induced obesity with IR mice and found ADR treatment enhanced glucose tolerance, ameliorates IR and promotes preferential use of carbohydrate over fat in fuel selection and indicated a negative relationship between ADR level and high blood glucose level and IR; thus supporting the findings of the current study.Thereafter, Tuna et al. (15) experimentally detected high serum ADR levels among animals received calorie-restricted diet, while was significantly lower among animals maintained on normal calorie intake, thus supporting the inverse correlation between serum ADR levels and calorie intake.
Moreover, in line with the results of the current study and against that of Dąbrowski et al. (47) multiple clinical trials approved the negative relation between ADR serum level and blood glucose and IR whereas Stevens et al. (49) reported that ADR levels in humans are sensitive to dietary macronutrients, perhaps due to habitual consumption of carbohydrate-rich diets suppressing circulating ADR levels.Zhang et al. (50) found IR index is a negative independent risk factor of ADR levels in obese adolescents.Yosaee et al. (51) reported that subjects with metabolic syndrome had significantly lower ADR levels and adropin/leptin ratio and ADR levels were correlated with metabolic syndrome and hence is a potentially protective agent against its development.

Table 1 :
-Demographic and clinical data determined at the 6 th week GA

Table 2 :
-Results of OGTT and HOMA-IR scoring of studied women at the 6 th and 24 th week GA th wk serum ADR levels showed negative significant correlation with BW, BMI, 6 th wk serum AFABP level and 2-hr PPBG, and with 24 th wk FBG, PPBG, FSI levels and HOMA-IR score.Also, 6 th wk serum AFABP levels showed positive significant correlation with age, 24 th and 6 th wk 2-hr PP blood glucose level, 24 th wk FBG and HOMA-IR score and 6 th wk and 24 th wk fasting serum insulin levels (Table3).Regression analysis of the

Table 4 :
-Regression analysis of baseline demographic data and laboratory findings in studied women as predictors for development of GDM at the 24 th wk GA Verification of baseline demographic and laboratory data using ROC curve analysis defined 6 th wk GA high serum AFABP and insulin, BMI and 2-hr PPBG, and low serum ADR, as significant predictors for development of IR at the 24 th wk GA.Regression analysis defined high serum insulin (β=0.411,p=0.0004) as positive and low serum ADR (β=-0.322,p=0.0007) as negative significant specific predictors for development of IR at the 24 th wk GA.