originl rticle Serum nesftin-1 levels re decresed in pregnnt women newly dignosed with gesttionl dibetes Esr Nur Ademoglu 1, Suheyl Gorr 2, Muge Keskin 3, Ayse Crlioglu 4, Rifki Ucler 5, Husmettin Erdmr 6, Cvit Culh 3, Ylcin Arl 3 ABSTRACT Objective: To investigte serum nesftin-1 levels t 24-28 weeks of pregnncy in women newly dignosed with gesttionl dibetes nd determine the ssocition of nesftin-1 with severl metbolic prmeters. Subjects nd methods: Forty women newly dignosed with gesttionl dibetes t 24-28 weeks of pregnncy nd 30 helthy pregnnt women mtched in ge nd gesttionl week were included in this cross-sectionl study. Serum nesftin-1 levels were nlyzed using ELISA, nd the reltionship between nesftin-1 nd severl metbolic prmeters were ssessed. Results: Serum nesftin-1 levels were found to be lower in women with gesttionl dibetes compred to the pregnnt women in the control smple (p = 0.020). Multiple liner regression nlysis reveled tht nesftin-1 ws lower in prticipnts with gesttionl dibetes independently from gesttionl ge, BMI, HOMA-IR, fsting plsm glucose, nd ge. In correltion nlysis, the only vrible tht ws found to hve sttisticlly significnt correltion with nesftin-1 ws gesttionl ge (p = 0.015, r = 0.30). Conclusion: Lower nesftin-1 levels in women with gesttionl dibetes compred to the control group t 24-28 weeks of gesttion drws ttention to nesftin-1 levels in gesttionl dibetes nd motivtes further reserch in this re. Arch Endocrinol Metb. 2017;61(5):455-9 Keywords Gesttionl dibetes; nesftin-1; insulin resistnce 1 Deprtment of Endocrinology nd Metbolism, Bolu University, Bolu, Turkey 2 Deprtment of Endocrinology nd Metbolism, Antly Eduction nd Reserch Hospitl, Antly, Turkey 3 Deprtment of Endocrinology nd Metbolism, Ankr Eduction nd Reserch Hospitl, Ankr, Turkey 4 Deprtment of Endocrinology nd Metbolism, Erzurum Eduction nd Reserch Hospitl, Erzurum, Turkey 5 Deprtment of Endocrinology nd Metbolism, Vn Yuzuncu Yil University Fculty of Medicine, Vn, Turkey 6 Deprtment of Biochemistry, Turgut Ozl University Fculty of Medicine, Ankr, Turkey Correspondence to: Suheyl Gorr Antly Eduction nd Reserch Hospitl, Deprtment of Endocrinology nd Metbolism sgorr@hotmil.com Received on June/17/2016 Accepted on July/4/2016 DOI: 10.1590/2359-3997000000288 INTRODUCTION Gesttionl dibetes mellitus (GDM) is defined s glucose intolernce of vrible degree tht begins or is first recognized during pregnncy. Worldwide, the incidence of GDM is grdully incresing every yer (1), vrying from 3% to 14%. Hyperglycemi during pregnncy is ssocited with the development of preeclmpsi, fetl mcrosomi, emergency cesren section, birth trum, nd neontl hypoglycemi. GDM is importnt not only due to its complictions but lso its conversion rtes to Type 2 dibetes mellitus (DM) s high s 2.6% to 70% over period of 6 weeks to 28 yers (2). Nesftin-1 is newly discovered hormone derived from nucleobindin-2 (NUCB2) nd hs been thought to be involved in the regultion of ppetite nd vrious metbolic conditions. It is prominently expressed in severl regions of the hypothlmus nd exists in the generl circultion. Nesftin-1 is lso produced in the peripherl tissues, including dipocytes, gstric mucos, nd pncretic bet-cells of both humns nd rts. One of the most importnt functions of nesftin-1 is reducing food intke. It cuses loss of ppetite, less frequent hunger, nd sense of fullness (3-6). It hs been demonstrted tht pncretic bet cells coloclize with nesftin/nucb2 in the islets of both mice nd rts, indicting the possible involvement of nesftin-1 in the regultion of insulin secretion from pncretic bet cells (7). It hs been shown tht fsting nesftin-1 levels were significntly lower in type 2 dibetic ptients, but its effects on those who hve newly been dignosed with GDM re unknown (8). In this study, we imed to exmine serum nesftin-1 concentrtions nd to investigte whether they hve ny correltion with insulin resistnce, nd to exmine 455
other metbolic prmeters in ptients who hd newly been dignosed with gesttionl dibetes. SUBJECTS AND METHODS Forty women newly dignosed with GDM t 24-28 weeks of pregnncy nd 30 helthy pregnnt women mtched in terms of ge nd gesttionl ge ws enrolled in the study. Dignosis of GDM ws bsed on 2015 Americn Dibetes Assocition recommendtions (9). A two-step pproch to screening with 1-h 50-g glucose lod test (GLT) followed by 3-h 100-g OGTT for those who screened positive ws used in exmining ll individuls. Pregnnt women who hd glucose levels higher thn 7.8 mmol/l one hour fter 50-g glucose-screening test were referred to 3-hour, 100-g orl glucose tolernce test nd Crpenter/Coustn thresholds were used. GDM ws dignosed if two or more of the following plsm glucose levels were exceeded: fsting 5.1 mmol/l, 1-hour 10 mmol/l, 2-hour 8.6 mmol/l, nd 3-hour 7.8 mmol/l (9). Exclusion criteri for the study were known pregesttionl dibetes, liver or renl dysfunction, cute or chronic inflmmtory diseses, pre-eclmpsi, use of nti-inflmmtory drugs, nd smoking. The body mss index ws clculted for ll prticipnts [BMI: body weight (kg)/squred height (m²)]. The ethicl committee of Ankr Eduction nd Reserch Hospitl pproved the study. A written informed consent ws obtined from ll the prticipnts. Blood smpling ws conducted in the morning following one night of fsting nd centrifuged immeditely. Serum smples were stored t 80ºC until the time of nlysis. Serum nesftin-1 concentrtions were nlyzed with ELISA kits from USCN Life Science Instruments (Wuhn, Chin). In this ssy system, the intr-ssy nd inter-ssy coefficient of vrition re lwys under 10%. This ssy employs the competitive inhibition enzyme immunossy technique. A monoclonl ntibody specific for humn NES1 ws pre-coted onto microplte. A competitive inhibition rection ws lunched between biotin-lbeled humn NES1 nd unlbeled humn NES1 (stndrds or smples) with the pre-coted ntibody specific for humn NES1. After incubtion, the unbound conjugte ws wshed off. Next, vidin conjugted to horserdish peroxidse (HRP) ws dded to ech microplte well nd incubted. The mount of bound HRP conjugte ws reverse proportionl to the concentrtion of NES1 in the smple. After ddition of the substrte solution, the intensity of color developed ws reverse proportionl to the concentrtion of NES1 in the smple. Serum glucose ws ssyed with hexokinse method using n Olympus AU 2700 nlyzer (Olympus UK, London, UK), nd hemoglobin A1c (HbA1c) ws mesured with high-performnce liquid chromtogrphy (HLC- 723 G7 HPLC systems; Tosoh Corportion., Tokyo, Jpn). Serum insulin ws determined using sndwich enzyme-linked immunosorbent ssy (ELISA) (Dynex DSX full utomtic ELISA nlyzer, USA). Insulin resistnce (IR) ws clculted for ech ptient using the homeostsis model ssessment insulin resistnce index (HOMA-IR), fsting plsm glucose (mmol/l) fsting insulin (miu/ml)/22.5. In ll sttisticl nlyses, SPSS softwre version 15.0 ws used. Distributions were tested for normlity using the Shpiro-Wilk test. The normlly distributed vribles were nlyzed with the Student s t-test, nd the dt vribles tht did not show norml distribution were compred with the Mnn-Whitney U-test. Bivrite correltions between nesftin-1 nd severl prmeters were nlyzed by Person correltion test for normlly distributed vribles. Spermn correltion test ws used for nonnormlly distributed vribles. Multiple regression nlysis ws used to exclude the possible confounding effect of other vribles on the result of ech correltion nlysis. P vlues less thn 0.05 were considered sttisticlly significnt for ll sttisticl nlyses. The dt for continuous vribles were presented s men ± stndrd devition. RESULTS Demogrphicl chrcteristics nd biochemicl vlues of women with GDM nd helthy controls re shown in Tble 1. The prticipnts with GDM nd the control group were similr in terms of men ge (29.6 ± 5.3 vs. 27.8 ± 6.0 yers, respectively). Serum fsting glucose, 1-h glucose fter 50-g glucose screening test, 1-h glucose, 2-h glucose, nd 3-h glucose fter 100-g orl glucose tolernce test, HbA1c, nd BMI were higher in women with GDM thn those in the control group (p < 0.05). Fsting insulin nd HOMA-IR were similr between the two groups. Serum nesftin-1 concentrtions were significntly found to be lower in the prticipnts with GDM compred to the controls (7.9 ± 2.8 vs. 11.2 ± 7.7 ng/ml, respectively, p = 0.020, Tble 1). 456
Tble 1. Demogrphicl chrcteristics nd biochemicl vlues of controls nd women with gesttionl dibetes Controls (n = 30) Gesttionl dibetes (n = 40) p vlue Age, yer 27.8 ± 6.0 29.6 ± 5.3 0.242 Gesttionl ge (week) 25.9 ± 1.5 26.2 ± 1.8 0.443 BMI (kg/m 2 ) 28.2 ± 1.5 31.0 ± 5.5 0.018 Fsting insulin (pmol/l) 55.2 ± 38.0 62.4 ± 38.2 0.441 HOMA-IR 1.4 ± 0.7 2.0 ± 1.7 0.074 50 g OGTT Glucose 1 h (mmol/l) 6.7 ± 1.7 10.3 ± 2.2 0.000 100 g OGTT Glucose 0 h (mmol/l) 4.0 ± 0.4 4.8 ± 0.8 0.001 Glucose 1 h (mmol/l) 9.1 ± 0.4 11 ± 1.4 0.000 Glucose 2 h (mmol/l) 7.6 ± 1.9 9.4 ± 2.3 0.000 Glucose 3 h (mmol/l) 5.7 ± 0.9 6.2 ± 2.2 0.004 HbA1c (mmol/mol) 32 36 0.004 (%) 4.8 ± 1.0 5.9 ± 1.1 Nesftin-1 (ng/ml) 11.2 ± 7.7 7.9 ± 2.8 0.020 BMI: body mss index; HOMA-IR: homeostsis model ssessment insulin resistnce index. The difference between controls nd gesttionl dibetes ws sttisticlly significnt (p < 0.05). Nesftin-1 (ng/ml) 50,00 40,00 30,00 20,00 10,00,00 * 11 3 Controls Gesttionl dibetes Figure 1. Distribution of serum nesftin-1 concentrtion in controls nd in gesttionl dibetes. 97 x 54 mm (300 x 300 DPI). Multiple liner regression nlysis reveled tht nesftin-1 ws lower in women with GDM independent of gesttionl ge, BMI, ge, fsting plsm glucose, nd HOMA-IR (Tble 2). Nesftin-1 levels hd sttisticlly significnt positive correltion with gesttionl ge (r = 0.30, p = 0.015, Tble 3). There ws not ny correltion between nesftin-1 nd BMI, HOMA-IR, fsting glucose, 1h-glucose fter 50-g glucose screening test, nd HbA1c (p > 0.05). Tble 2. Multiple liner regression nlysis including nesftin-1, gesttionl ge, BMI, ge, fsting plsm glucose, nd HOMA-IR Stndrdized coefficient (β) p vlue Gesttionl ge 0.021 0.553 Age 0.014 0.197 BMI 0.032 0.048 Nesftin-1-0.026 0.014 HOMA-IR -0.038 0.423 Fsting plsm glucose 0.008 0.054 β coefficients nd p vlues re given. Dependent vrible; gesttionl dibetes. Independent vribles; nesftin-1, gesttionl ge, BMI (body mss index), ge, fsting plsm glucose, nd HOMA-IR (homeostsis model ssessment insulin resistnce index). Sttisticlly significnt (p < 0.05). Tble 3. The correltion nlyses of nesftin-1 with some prmeters in women with gesttionl dibetes p vlue Age 0.19 0.09 BMI 0.46 0.12 Fsting insulin 0.14 0.25 Fsting blood glucose 0.80-0.04 HOMA-IR 0.22-0.21 1-h glucose (50 g OGTT) 0.18-0.23 HbA1c 0.308-0.13 Gesttionl ge 0.015 0.30 BMI: body mss index; HOMA-IR: homeostsis model ssessment index. The correltion between nesftin 1 nd gesttionl ge ws sttisticlly significnt in gesttionl dibetes (p < 0.05). DISCUSSION In the present study, we demonstrted tht serum nesftin-1 levels t 24-28 weeks of gesttion re significntly lower in women newly dignosed with GDM compred to helthy pregnnt women. In 2010, Aydın S. first described the presence of nesftin-1 in brest milk (10). In tht study, nesftin-1 ws investigted in serum, milk, nd colostrum of lctting women who were dignosed with GDM nd in helthy lctting women. Nesftin-1 ws demonstrted to be lower in the serum of lctting women dignosed with GDM thn tht of the control prticipnts. It ws suggested tht the brest tissue ws likely to be source of nesftin-1 just like the centrl nervous system, dipocytes, gstric endocrine cells, nd pncretic bet cells. It ws lso pointed out tht this my thus be importnt for growth, energy regultion, nd mturtion of the gstrointestinl system in neontes. r 457
Clinicl significnce of nesftin-1 in vrious metbolic diseses like obesity, Type 2 DM, nd insulin resistnce ws demonstrted in severl studies in the literture, but its effects on GDM re unknown. The literture reports only one study bout the nesftin-1 levels in gesttionl dibetes. In this study, Asln nd cols. ssessed both serum nd cord blood pelin nd nesftin-1 levels in pregnnt women with GDM nd in helthy pregnnt women (11). Cord blood nesftin-1 nd pelin-36 levels were comprble between women with GDM nd controls, serum pelin-36 concentrtions were found to be higher, nd serum nesftin-1 concentrtions were found to be lower in women with GDM compred to controls. The designs differ between our study nd tht study in some spects, including the time of nesftin-1 smpling nd ptient selection. In the study of Asln nd cols., the study group ws composed of pregnnt women who were being followed with dignosis of GDM nd whose blood glucose levels were strictly controlled, wheres our study included pregnnt women who were newly dignosed with GDM so tht none of them were on restricted diets or using insulin therpy. Additionlly, Asln nd cols. did not point out whether the women dignosed with GDM were using n insulin regimen or only following specific diet. They mesured nesftin-1 levels t the time of birth before plcent delivery in ptients with GDM nd in controls. Differently, we mesured nesftin-1 t 24-28 weeks of pregnncy in control nd study groups. In 2012, Boutsikou nd cols. mesured cord blood nesftin-1 nd insulin concentrtions in 40 lrge (9 born from dibetic nd 31 from non-dibetic mothers) nd 20 pproprite for gesttionl ge pregnncies (12). Cord blood nesftin-1 concentrtions were shown to be significntly lower in lrge gesttionl ge pregnncies compred to pproprite for gesttionl ge pregnncies. Furthermore, fetl nesftin-1 concentrtions were found to be elevted in infnts born from mothers with GDM compred to those born from mothers without GDM. Serum nesftin-1 levels in obesity nd Type 2 DM reported in the literture ws rther controversil. In 2010, Tsuchiy nd cols. reported tht fsting concentrtions of nesftin-1 were significntly lower in persons with high BMI compred to non-obese prticipnts (13). However, in contrst to this study, Rmnjney nd cols. found incresed nesftin-1 levels in obese sttes in both rodents nd humns in their study (14). In recent study, Gonzlez nd cols. demonstrted tht nesftin-1/nucb2 mrna expression in the pncretic islets is mrkedly incresed in Goto-Kkizki rts, model with Type 2 DM, chrcterized by impired insulin secretion nd viscerl ft ccumultion (15). Involvement of nesftin-1 in the regultion of insulin secretion remins lrgely unknown. In 2013, Nkt nd cols. reported tht nesftin-1 exerts its metbolic effects prtly vi promoting insulin relese by C +2 influx through L-Type C +2 chnnels independently of protein kinse A (PKA) nd phospholipse A 2 (PLA 2 ) so tht dysregultion of nesftin-1 might be implicted in metbolic disorders, prticulrly in Type 2 DM (16). In 2010, Su nd cols. reported tht intrvenous injection of nesftin-1 suppresses hyperglycemi in ob/ ob mice by enhncing insulin secretion (17). In nother study, Li nd cols. demonstrted tht fsting nesftin-1 levels were found to be decresed in persons with Type 2 DM compred to helthy prticipnts nd persons with Type 1 DM (8). Additionlly, it ws pointed out tht lthough this result is uncler, nesftin-1 my be cusl fctor in dibetic hyperphgi. In 2012, in contrst to the study of Li nd cols., Zhng nd cols. demonstrted elevted levels of nesftin-1 in prticipnts with Type 2 DM nd with impired glucose tolernce (IGT) compred to controls (18). It ws shown tht plsm nesftin-1 ws positively correlted with BMI, HbA1c, fsting blood glucose, fsting plsm insulin, nd HOMA-IR. In recent study, Ding nd cols. reveled tht Type 2 DM ptients with peripherl rteril disese (PAD) exhibited mrked lower serum nesftin-1 concentrtions thn those without PAD. They hypothesized tht serum nesftin-1 levels were inversely correlted with the development nd severity of PAD in Type 2 DM ptients (19). Previous studies revel conflicting dt bout the correltions of nesftin-1 with metbolic prmeters. For instnce, Zhng nd cols. showed tht nesftin-1 ws positively correlted with BMI, HbA1c, fsting blood glucose, fsting insulin, nd HOMA-IR in prticipnts with newly dignosed Type 2 DM (18). In contrst to tht study, Deniz nd cols. reveled negtive correltion between nesftin-1 nd BMI, fsting blood glucose, nd HOMA-IR in ptients with polycystic ovry syndrome, n endocrine disorder commonly presenting with obesity nd insulin resistnce s well s hyperndrogenemi nd hirsutism (20). Ding nd cols. lso reveled negtive correltion between nesftin-1 nd BMI in their study 458
(19). In the present study, we did not find ny correltion between nesftin-1 nd fsting glucose, 1-h glucose fter 50-g glucose screening test, 1-h, 2-h, nd 3-h glucose fter 100-g orl glucose tolernce test, HbA1c, HOMA-IR, nd BMI. We found significnt positive correltion between nesftin-1 nd gesttionl ge, contrry to the results of the study by Asln nd cols., s they hd observed negtive correltion between nesftin-1 nd gesttionl ge (11). Becuse few dt re currently present in the literture, the circulting levels of nesftin-1 nd its correltion with severl prmeters in GDM re not well-known nd might require further investigtion. Mesurement of serum nesftin-1 consecutively during the gesttion my provide more evidence bout the role of nesftin-1 in GDM in future reserch. In conclusion, we hve demonstrted for the first time reduced nesftin-1 levels t 24-28 weeks of pregnncy in women newly dignosed with gesttionl dibetes. Although the significnce of this result is yet uncler, it is importnt becuse it drws ttention to nesftin-1 levels in GDM nd my shed light on further reserch in this re. Funding sttement nd ny grnts or fellowships: nothing. Disclosure: no potentil conflict of interest relevnt to this rticle ws reported. REFERENCES 1. Metzger BE, Gbbe SG, Persson B, Buchnn TA, Ctlno PA, Dmm P, et l. Interntionl ssocition of dibetes nd pregnncy study group recommendtions on the dignosis nd clssifiction of hyperglycemi in pregnncy. Dibetes Cre. 2010;33:676-82. 2. Kim C, Newton KM, Knopp RH. Gesttionl dibetes nd the incidence of type-2 dibetes: systemtic review. Dibetes Cre. 2002;25:1862-8. 3. Oh-I S, Shimizu H, Stoh T, Okd S, Adchi S, Inoue K, et l. Identifiction of nesftin-1 s stiety molecule in the hypothlmus. Nture. 2006;443:709-12. 4. Stengel A, Goebel M, Wng L, Rivier J, Kobelt P, Mönnikes H, et l. Centrl nesftin-1 reduces drk-phse food intke nd gstric emptying in rts: differentil role of corticotropin-relesing fctor 2 receptor. Endocrinology. 2009;150:491-9. 5. Foo KS, Bruner H, Ostenson CG, Broberger C. Nucleobindin-2/ nesftin in the endocrine pncres: distribution nd reltionship to glycemic stte. J Endocrinol. 2010;204:255-63. 6. Stengel A, Tche Y. Nesftin-1 role s possible new potent regultor of food intke. Regul Pept. 2010;163:18-23. 7. Gonzlez R, Tiwri A, Unnippn S. Pncretic bet cells coloclize insulin nd pronesftin immunorectivity in rodents. Biochem Biophys Res Commun. 2009;381:643-8. 8. Li QC, Wng HY, Chen X, Gun HZ, Jing ZY. Fsting plsm levels of nesftin-1 in ptients with Type 1 nd Type 2 dibetes mellitus nd the nutrient-relted fluctution of nesftin-1 level in norml humns. Regul Pept. 2010;159:72-7. 9. Stndrds of medicl cre in dibetes--2015: summry of revisions. Dibetes Cre. 2015;38 Suppl:S4. 10. Aydın S. The presence of the peptides pelin, ghrelin nd nesftin-1 in the humn brest milk, nd the lowering of their levels in ptients with gesttionl dibetes mellitus. Peptides. 2010;31:2236-40. 11. Asln M, Celik O, Celik N, Turkcuoglu I, Yılmz E, Krer A, et l. Cord blood nesftin-1 nd pelin-36 levels in gesttionl dibetes mellitus. Endocrine. 2012;41:424-9. 12. Boutsikou T, Brin DD, Boutsikou M, Kflidis G, Pitopoulou D, Bk S, et l. Cord blood nesftin-1 in lrge for gesttionl ge pregnncies. Cytokine. 2013;61:591-4. 13. Tsuchiy T, Shimizu H, Ymd M, Oski A, Oh-I S, Ariym Y, et l. Fsting concentrtions of nesftin-1 re negtively correlted with body mss index in non-obese mles. Clin Endocrinol. 2010;73:484-90. 14. Rmnjney M, Chen J, Brown JE, Hllschmid M, Ptel S, Kern W, et l. Identifiction of nesftin-1 in humn nd murine dipose tissue: novel depot-specific dipokine with incresed levels in obesity. Endocrinology. 2010;151:3169-80. 15. Gonzlez R, Reingold BK, Go X, Gidhu MP, Tsushim RG, Unnippn S. Nesftin-1 exerts direct, glucose-dependent insulinotropic ction on mouse islet β- nd MIN6 cells. J Endocrinol. 2011;208:R9-16. 16. Nkt M, Mnk K, Ymmoto S, Mori M, Yd T. Nesftin-1 enhnces glucose-induced insulin secretion by promoting C2+ influx through L-Type chnnels in Mouse islet β-cells. Endocrine J. 2011;58:305-13. 17. Su Y, Zhng J, Tng Y, Bi F, Liu JN. The novel function of nesftin-1: nti-hyperglycemi. Biochem Biophys Res Commun. 2010;391:1039-42. 18. Zhng Z, Li L, Yng M, Liu H, Boden G, Yng G, et l. Incresed Plsm Levels of Nesftin-1 in Ptients with Newly Dignosed Type 2 Dibetes Mellitus. Exp Clin Endocrinol Dibetes. 2012;120:91-5. 19. Ding S, Qu W, Dng S, Xie X, Xu J, Wng Y, et l. Serum nesftin-1 is reduced in type 2 dibetes mellitus ptients with peripherl rteril disese. Med Sci Monit. 2015;21:987-91. 20. Deniz R, Gurtes B, Aydin S, Celik H, Shin I, Bykus Y, et l. Nesftin-1 nd other hormone ltertions in polycystic ovry syndrome. Endocrine. 2012;42:694-9. 459