褪黑素受体1B基因多态性及其对葡萄糖转运的调控在妊娠期糖尿病中的作用

Role of melatonin receptor 1B gene polymorphism and its effect on the regulation of glucose transport in gestational diabetes mellitus 褪黑素受体1B基因多态性及其对葡萄糖转运的调控在妊娠期糖尿病中的作用 1 first-author 1 1 1 1 1 1 1 1 1 2 3 1 1 1 1 corresp Jun YU, yjtj2010@163.com Jun YU, yjtj2010@163.com yjtj2010@163.com https://orcid.org/0000-0003-4718-409X 华中科技大学同济医学院附属同济医院妇产科， 中国武汉， 430030 华中科技大学同济医学院附属同济医院妇产科， 中国武汉， 430030 Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 南昌大学附属第一医院妇产科， 中国南昌， 330006 南昌大学附属第一医院妇产科， 中国南昌， 330006 Department of Obstetrics and Gynecology, the First Affiliated Hospital of Nanchang University, Nanchang 330006, China Department of Obstetrics and Gynecology, the First Affiliated Hospital of Nanchang University, Nanchang 330006, China 亚琛工业大学医院妇产科， 德国亚琛， 52074 亚琛工业大学医院妇产科， 德国亚琛， 52074 Department of Obstetrics and Gynecology, University Hospital Aachen, RWTH Aachen University, Aachen 52074, Germany Department of Obstetrics and Gynecology, University Hospital Aachen, RWTH Aachen University, Aachen 52074, Germany Melatonin receptor 1B (MT2, encoded by the MTNR1B gene), a high-affinity receptor for melatonin, is associated with glucose homeostasis including glucose uptake and transport. The rs10830963 variant in the MTNR1B gene is linked to glucose metabolism disorders including gestational diabetes mellitus (GDM); however, the relationship between MT2-mediated melatonin signaling and a high birth weight of GDM infants from maternal glucose abnormality remains poorly understood. This article aims to investigate the relationship between rs10830963 variants and GDM development, as well as the effects of MT2 receptor on glucose uptake and transport in trophoblasts. TaqMan-MGB (minor groove binder) probe quantitative real-time polymerase chain reaction (qPCR) assays were used for rs10930963 genotyping. MT2 expression in the placenta of GDM and normal pregnant women was detected by immunofluorescence, western blot, and qPCR. The relationship between MT2 and glucose transporters (GLUTs) or peroxisome proliferator-activated receptor γ (PPARγ) was established by western blot, and glucose consumption of trophoblasts was measured by a glucose assay kit. The results showed that the genotype and allele frequencies of rs10830963 were significantly different between GDM and normal pregnant women ( P < 0.05). The fasting, 1-h and 2-h plasma glucose levels of G-allele carriers were significantly higher than those of C-allele carriers ( P < 0.05). Besides, the protein and messenger RNA (mRNA) expression of MT2 in the placenta of GDM was significantly higher than that of normal pregnant women ( P < 0.05). Melatonin could stimulate glucose uptake and GLUT4 and PPARγ protein expression in trophoblasts, which could be attenuated by MT2 receptor knockdown. In conclusion, the rs10830963 variant was associated with an increased risk of GDM. The MT2 receptor is essential for melatonin to raise glucose uptake and transport, which may be mediated by PPARγ. 目的 2 探讨褪黑素受体1B基因（ MTNR1B ）rs10830963位点多态性与妊娠期糖尿病（GDM）发生的关联性，以及MT2受体（由 MTNR1B 基因编码）在滋养细胞葡萄糖摄取和转运过程中的作用及机制。创新点： 本研究探讨了中国中部地区女性人群 MTNR1B 基因rs10830963位点多态性与GDM发生发展之间的关系，以及MT2受体对滋养细胞中葡萄糖摄取和转运的影响。以此在一定程度上揭示MT2受体介导的褪黑素信号与GDM孕妇的新生儿高出生体重之间的关联性及可能的作用机制。 方法 2 本研究采用TaqMan-MGB荧光定量聚合酶链反应（qPCR）对rs10930963位点进行基因分型，分析该位点突变与GDM发生及相关代谢指标异常的关联性。采用免疫荧光、蛋白质印迹法（western blot）和qPCR检测GDM和正常孕妇胎盘中MT2受体的表达水平。体外培养滋养细胞系HTR-8/SVneo并给予褪黑素处理，利用western blot检测褪黑素信号对滋养细胞MT2受体、葡萄糖转运体（GLUTs）和激活过氧化物酶增殖物激活受体γ（PPARγ）蛋白表达水平的影响，葡萄糖检测试剂盒检测其对滋养细胞葡萄糖消耗水平的影响。进一步利用小干扰RNA（siRNA）敲低MT2受体，并采用western blot和葡萄糖检测试剂盒分别检测滋养细胞GLUTs和PPARγ的蛋白表达水平和葡萄糖消耗水平，以此探究MT2受体在褪黑素调控葡萄糖摄取和转运中的作用。 结论 2 MTNR1B 基因的rs10830963位点多态性与GDM发生风险增加有关。MT2受体是褪黑素促进葡萄糖摄取和转运的关键受体，该过程可能由PPARγ介导。 妊娠期糖尿病（GDM） 褪黑素受体 1B（ MTNR1B ） 单核苷酸多态性（SNP） 葡萄糖摄取 葡萄糖转运体（GLUTs） 激活过氧化物酶增殖物激活受体γ（PPARγ） Gestational diabetes mellitus (GDM) Melatonin receptor 1B ( MTNR1B ) Single nucleotide polymorphism (SNP) Glucose uptake Glucose transporters (GLUTs) Peroxisome proliferator-activated receptor γ (PPARγ) 1 Introduction 1 Gestational diabetes mellitus (GDM) is a metabolic disorder characterized by abnormal glucose homeostasis, affecting more than one out of seven pregnant women worldwide ( Wu et al., 2021 Wu et al., 2021 ; Zhu et al., 2021 Zhu et al., 2021 ). GDM is associated with several adverse outcomes for the mother (preeclampsia, cesarean section, metabolic disorders, etc.) and the fetus (macrosomia, neonatal hypoglycemia, metabolic syndrome, etc.) ( Pettitt et al., 1985 Pettitt et al., 1985 ; The HAPO Study Cooperative Research Group, 2008 The HAPO Study Cooperative Research Group, 2008 ; Bellamy et al., 2009 Bellamy et al., 2009 ; Fadl et al., 2014 Fadl et al., 2014 ; Alharbi et al., 2022 Alharbi et al., 2022 ). The pathogenesis of GDM is generally thought to be the result of a combination of genetic and environmental factors ( Feng et al., 2019 Feng et al., 2019 ). Since GDM and type 2 diabetes mellitus (T2DM) share similar pathological characteristics of β‍-cell dysfunction, insulin resistance, and abnormal glucose uptake and transport, GDM is also likely to be a polygenic disease ( Khan et al., 2014 Khan et al., 2014 ; Powe et al., 2018 Powe et al., 2018 ). Melatonin receptor 1B (MT2, encoded by the MTNR1B gene) is a member of the G protein-coupled receptor family. In the past ten years, genome-wide association studies (GWAS) had determined that the variations of MTNR1B were closely related to elevated fasting plasma glucose (FPG) and the increased occurrence of T2DM ( Karamitri and Jockers, 2019 Karamitri and Jockers, 2019 ). Moreover, many common single nucleotide polymorphism (SNP) loci of MTNR1B gene have been revealed as risk factors of T2DM by several studies ( Ling et al., 2011 Ling et al., 2011 ; Patel et al., 2018 Patel et al., 2018 ; Shen and Jin, 2019 Shen and Jin, 2019 ). Among them, the rs10830963 locus has been widely investigated, and the results indicated that the variation of rs10830963 is a risk factor for T2DM. In addition, rs10830963 variants were also associated with elevated FPG ( Karamitri and Jockers, 2019 Karamitri and Jockers, 2019 ). It is suggested that rs10830963 SNP may be closely associated with glucose metabolism. Considering that abnormal glucose metabolism is one of the pathogenesis factors of GDM, it is necessary to determine its genetic background. MT2 receptor displays high affinity for melatonin (MLT), a hormone primarily synthesized and released from the pineal gland and other sites of the body, including placental tissue ( Soliman et al., 2015 Soliman et al., 2015 ). In addition to its important role in regulating circadian rhythms, MLT has also been identified as a key regulator of glucose metabolism. For instance, in a rat model of smoking-induced hyperglycemia, MLT preserves insulin secretion and hepatic glycogen synthesis mediated by the MT2 receptor ( Li et al., 2018 Li et al., 2018 ). The analysis of overexpressed MT2 receptor combined with MLT in skeletal muscle cells indicated the vital role of MT2 receptor for MLT signaling in regulating glucose uptake through activating insulin receptor substrate-1 (IRS-1)/phosphatidylinositol 3-kinase (PI3K) activity ( Ha et al., 2006 Ha et al., 2006 ). Thus far, studies on the effect of MLT on the regulation of glucose homeostasis have predominantly focused on pancreatic islets, skeletal muscle, and adipose tissue ( Owino et al., 2019 Owino et al., 2019 ; Guan et al., 2021 Guan et al., 2021 ), and research on the effect of MLT on the placenta is scarce. However, the placenta is an important fetal organ during pregnancy, playing a crucial role in regulating maternal and fetal glucose homeostasis. In GDM, maternal hyperglycemia leads to excess blood glucose passing through the placenta, resulting in fetal hyperglycemia. This causes the fetal pancreas to secrete more insulin to eliminate blood glucose, and leads to the storage of excess energy in the form of fat ( ACOG, 2020 ACOG, 2020 ). Therefore, exploring the effect of MLT signaling on glucose transport in the placenta is of particularly importance to understand the impact of maternal GDM on the offspring’s glucose metabolism. Glucose transporters (GLUTs) are facilitative transport proteins with a function to translocate glucose across the plasma membrane, and the expression of GLUT1, GLUT3, and GLUT4 has been previously described in the placenta ( Borges et al., 2019 Borges et al., 2019 ; James-Allan et al., 2019 James-Allan et al., 2019 ; Zhang et al., 2021 Zhang et al., 2021 ). GLUTs are located in the syncytiotrophoblast of the placenta, including the maternal-facing microvillous membrane (MVM) and the fetal-facing basal membrane (BM). Net glucose transfer primarily depends on the difference in glucose concentration between the maternal and fetal sides of the placenta, the metabolic level of placental cells, and the expression levels of GLUTs. Notably, placental glucose transport is mainly determined by the intrauterine glucose level and the expression and activity of GLUTs in MVM and BM, which play roles in glucose uptake and glucose transport, respectively ( Castillo-Castrejon and Powell, 2017 Castillo-Castrejon and Powell, 2017 ). GLUTs are regulated by multiple signaling pathways. In peroxisome proliferator-activated receptor γ (PPARγ)‍-deficient 3T3-L1 adipocytes, glucose uptake and the expression levels of GLUT1 and GLUT4 were decreased, suggesting the important role of PPARγ in regulating glucose uptake and transport ( Beaumont et al., 2018 Beaumont et al., 2018 ). However, the effect of MLT signaling on regulating glucose uptake in the placenta remains unclear. Herein, we set out to assess the relationship between rs10830963 variants in the MTNR1B gene and the development of GDM. Then, we further explored the effect of MT2 receptor on the regulation of glucose transport in the presence of MLT in the placenta. Thus, novel targets can be revealed for the prevention of abnormal glucose metabolism in offspring born to mothers with GDM. 2 Results 1 2.1 Clinical and laboratory parameters of pregnant women 2 In total, we recruited 856 pregnant women for this study. The mean age of 320 pregnant women in the GDM group was (32.83±4.79) years, while that of 536 pregnant women in the normal pregnancy (NP) group was (30.32±4.07) years. Compared with the NP group, the GDM group had significantly higher age, pre-pregnancy body mass index (BMI), plasma glucose, and total cholesterol (TC) level ( P < 0.05). However, pregnant women with GDM had significantly lower plasma triglyceride (TG) level than the NP group ( P < 0.05; Table 1 Table 1 ). GDM: gestational diabetes mellitus; NP: normal pregnancy; BMI: body mass index; FPG: fasting plasma glucose; OGTT: oral glucose tolerance test; TG: triglyceride; TC: total cholesterol. 1 mmHg=0.133 kPa. All data are presented with mean±standard deviation (SD). * P < 0.05. Parameter NP ( n =536) GDM ( n =320) P value Gestational age (years) 30.32±4.07 32.83±4.79 < 0.001 * Pre-pregnancy BMI (kg/m 2 ) 20.76±2.12 21.35±2.44 < 0.001 * Weight-gain during pregnancy (kg) 16.65±0.61 14.41±0.62 0.012 * Systolic pressure (mmHg) 117.22±11.23 117.58±11.66 0.621 Diastolic pressure (mmHg) 75.71±8.33 75.82±8.02 0.350 FPG OGTT (mmol/L) 4.49±0.30 5.11±0.71 < 0.0001 * 1-h OGTT (mmol/L) 7.41±1.29 9.73±1.85 < 0.0001 * 2-h OGTT (mmol/L) 6.54±1.00 8.55±1.57 < 0.0001 * TG (mmol/L) 3.19±0.16 3.94±0.30 0.022 * TC (mmol/L) 5.87±1.18 5.62±1.09 0.001 * 2.2 MTNR1B gene polymorphism of pregnant women 2 The distribution of genotype frequencies in healthy pregnant women was in compliance with the Hardy-Weinberg equilibrium (HWE) ( P =0.732). The frequency of CG genotype was the highest compared to CC and GG genotypes (53% for GDM group, 48% for NP group). The frequency of the CC genotype was lower than that of the GG genotype in the GDM group (CC=21%, GG=25%), while that of the CC genotype was higher than that of the GG genotype in the NP group (CC=33%, GG=19%). The frequency of the G allele was significantly higher than others in the NP group (52% for GDM group, 43% for NP group; P < 0.05). Moreover, using the three genotype models (dominant model, recessive model, and additive model) for pregnant women, we could find a significant relationship between rs10830963 and the risk of GDM after adjusting for age and BMI ( P < 0.05). The odd ratios for the three genotype models were as follows: 1.818 (confidence interval (CI): 1.290–2.562) for the dominant model, 1.459 (CI: 1.024–1.648) for the recessive model, and 2.085 (CI: 1.355–3.209) for the addictive model ( Table 2 Table 2 ). # Data are expressed as number (percentage). * P values were adjusted for age and body mass index; ** P value for allelic gene. GDM: gestational diabetes mellitus; NP: normal pregnancy; OR: odd ratio; CI: confidence interval; HWE: Hardy-Weinberg equilibrium. rs10830963 NP ( n =536) # GDM ( n =320) # OR (95% CI) P value HWE Allele CC 175 (33%) 68 (21%) 0.732 CG 259 (48%) 171 (53%) GG 102 (19%) 81 (25%) C 609 (57%) 307 (48%) G 463 (43%) 333 (52%) 1.427 (1.413–1.441) 0.000 ** Genotype model Dominant 1.818 (1.290–2.562) 0.001 * Recessive 1.459 (1.024–1.648) 0.036 * Additive 2.085 (1.355–3.209) 0.001 * 2.3 Relationships between the genotype frequencies of rs10830963 and the metabolic parameters of pregnant women 2 We explored the relationship between the variation of rs10830963 genotype distribution and the metabolic parameters of pregnant women. The results showed a significant association of plasma glucose level with rs10830963 genotype distribution, especially for the FPG level ( P < 0.001 in dominant, recessive, and additive models). Furthermore, the distribution of rs10830963 genotype was also significantly associated with the 1-h ( P < 0.05 in dominant, recessive and additive models) and 2-h plasma glucose levels ( P < 0.05 in additive model) after drinking the glucose solution ( Table 3 Table 3 ). FPG: fasting plasma glucose; OGTT: oral glucose tolerance test; TG: triglyceride; TC: total cholesterol. All data are presented as mean±standard deviation (SD). P values were adjusted for age and body mass index. 2 Parameter 3 Genotype 3 P value CC ( n =243) GC ( n =430) GG ( n =183) Dominant model Recessive model Additive model Weight-gain during pregnancy (kg) 15.32±3.38 15.17±4.54 14.64±4.24 0.895 0.574 0.854 FPG OGTT (mmol/L) 4.63±0.50 4.78±0.63 4.91±0.62 0.000 0.000 0.000 1-h OGTT (mmol/L) 8.18±1.85 8.45±1.99 8.76±1.81 0.029 0.011 0.003 2-h OGTT (mmol/L) 7.26±1.65 7.41±1.59 7.64±1.65 0.144 0.060 0.033 TG (mmol/L) 4.16±1.89 4.49±3.46 3.31±1.04 0.325 0.319 0.235 TC (mmol/L) 5.79±1.08 5.83±1.16 5.71±1.09 0.577 0.211 0.262 Neonatal birthweight (kg) 3.29±0.40 3.23±0.38 3.27±0.42 0.232 0.644 0.156 Neonatal plasma glucose (mmol/L) 3.14±1.28 3.24±1.29 3.10±1.02 0.391 0.622 0.285 2.4 Expression of MT2 receptor in placental tissues 2 In order to explore whether the MT2 receptor was expressed in the placenta, our study measured the protein expression of this receptor in the placental tissues by immunofluorescence. Co-localization by immunofluorescence was used to identify the expression of MT2 (red) in the trophoblast, marked by cytokeratin 7 (CK7, green). We observed that MT2 staining was strongly positive in CK7-positive cells, and MT2 expression was markedly increased in the placental tissue of the GDM group (Figs. 1 1 a– 1 1 d). Furthermore, western blot showed that MT2 expression was significantly elevated in the GDM group, as compared with the NP group (Figs. 1 1 e and 1 1 f). MTNR1B messenger RNA (mRNA) expression in the GDM group was also significantly higher than that in the NP group ( Fig. 1 Fig. 1 g). 2.5 Effects of MLT on the expression of GLUTs, MT2 receptor, and PPARγ 2 In order to understand the effect of MLT signaling on glucose uptake and transport, HTR-8/SVneo cells were treated with MLT (0.25, 0.5, and 1.0 mmol/L) or received no treatment (dimethyl sulfoxide (DMSO)). Quantification showed that glucose consumption was significantly increased in MLT-treated (0.25, 0.5, and 1.0 mmol/L) cells compared with the control group ( Fig. 2 Fig. 2 a). Moreover, GLUT4 protein expression also significantly increased after incubation with 1.0 mmol/L MLT compared with the control group. However, for the GLUT1 and GLUT3 transporters, no significant change in protein expression could be observed (Figs. 2 2 b and 2 2 d). In order to explore the mechanism of MLT in regulating glucose uptake and transport, we measured the expression of MT2, a high-affinity receptor for MLT and PPARγ, which is key regulator of glucose uptake and transport. We found that the expression levels of MT2 and PPARγ proteins were significantly increased after incubation with 1.0 mmol/L MLT compared with the control group (Figs. 2 2 c and 2 2 e). Taken together, these findings suggested that the MLT-induced increases in glucose uptake and transport are likely due to the MT2 receptors and PPARγ. 2.6 Inhibition effects of MLT signaling on glucose transport and PPARγ expression by knockdown of the MT2 receptor 2 In order to determine whether MT2 is a key receptor mediating MLT signaling in the regulation of glucose uptake and transport, HTR-8/SVneo cells were transfected with small interfering RNAs (siRNAs) (si-MT2 and si-NC) in order to silence the expression of MT2 receptor combined with 1.0 mmol/L MLT ( Fig. 3 Fig. 3 a). We analyzed the effectiveness of the three siRNAs (siRNA1, siRNA2, and siRNA3), and siRNA3 was the most effective in silencing MT2 expression (Figs. 3 3 b and 3 3 c). Then, we measured the alterations of glucose uptake in cells and the expression of GLUTs. We observed that the glucose consumption of si-MT2+1.0 mmol/L MLT group was significantly decreased compared with the si-NC+1.0 mmol/L MLT group ( Fig. 3 Fig. 3 d). Furthermore, the expression levels of GLUT1 and GLUT4 were also significantly decreased in the si-MT2+1.0 mmol/L MLT group compared with the si-NC+1.0 mmol/L MLT group (Figs. 3 3 e and 3 3 f). With the aim to further confirm that transcription factor PPARγ acts as a downstream of MT2 receptor in the regulation of GLUTs, we detected PPARγ protein expression. We observed that it was significantly decreased in the si-MT2+1.0 mmol/L MLT group compared with the si-NC+1.0 mmol/L MLT group (Figs. 3 3 g and 3 3 h). Overall, these findings suggested that the function of MLT signaling on the upregulation of glucose uptake and transport is mediated by MT2 receptors. 3 Discussion 1 This study revealed that the rs10830963 variant was significantly associated with GDM, as well as elevated FPG, 1-h and 2-h plasma glucose levels. Studies in Saudi, Mexican Americans, and Greek populations also identified a relationship between the rs10830963 variant and GDM susceptibility ( Vlassi et al., 2012 Vlassi et al., 2012 ; Ren et al., 2014 Ren et al., 2014 ; Alharbi et al., 2019 Alharbi et al., 2019 ). Besides, the rs10830963 variant was also associated with offspring’s birth weight ( Beaumont et al., 2018 Beaumont et al., 2018 ). The variant of another locus of MTNR1B gene, rs10830962, significantly interacts with the offspring’s obesity ( Liang et al., 2020 Liang et al., 2020 ). Notably, some studies proposed that the effect of maternal genotype on fetal metabolic disorders such as high birth weight is due to the high glucose intrauterine environment, rather than through shared alleles with the fetus ( Beaumont et al., 2018 Beaumont et al., 2018 ; Zhou et al., 2022 Zhou et al., 2022 ). Raised maternal glucose leads to elevated placental glucose transfer and fetal insulin secretion, resulting in higher offspring’s birth weight ( Haggarty et al., 2002 Haggarty et al., 2002 ). Therefore, understanding the maternal determinants of fetal growth could be beneficial for reducing the prevalence of metabolic disorders in offspring. We found that carrying the rs10830963 G risk allele significantly increased the occurrence of GDM, as compared with carrying the C risk allele. The rs10830963 is localized in the 5' promoter region of the MTNR1B gene, which may result in alterations of mRNA and protein expression ( Karamitri and Jockers, 2019 Karamitri and Jockers, 2019 ). In the placenta of GDM, the expression of MT2 protein in the carriers of the G allele was also significantly higher than that in the carriers of the C allele ( Li et al., 2019 Li et al., 2019 ), suggesting that the variant of rs10830963 may contribute to the development of GDM by altering of transcription and protein expression of MTNR1B. In this study, elevated expression levels of MTNR1B mRNA and protein were observed in GDM placenta, and this difference was predominantly exhibited in trophoblast cells. Therefore, we assumed that the increased fetal glucose uptake may be related to the alteration of MT2 receptor expression in the placenta. MT2 is a high-affinity receptor for MLT expressed at central and peripheral sites. Despite the low levels of MT2 expression in peripheral tissues, direct effects of MLT on specific tissue functions, such as glucose uptake and transport, glycogen synthesis, and glycolysis, have been reported ( Ha et al., 2006 Ha et al., 2006 ; Li et al., 2018 Li et al., 2018 ). In this study, we found that MLT upregulated the uptake and transport of glucose in HTR-8/SVneo cells via the MT2 receptor. The placenta mediates maternal-fetal nutrient transport, and the transportation of glucose provides energy for fetal growth and metabolism. However, extra glucose transferring to fetus may lead to fetal hyperglycemia or macrosomia. Thus, we speculated that the MLT signaling pathway mediated by the MT2 receptor may be involved in the mechanism of fetal hyperglycemia and macrosomia in GDM. The transportation of glucose in the placenta is mediated by GLUTs. GLUT1 is a universal transporter with approximately 3-fold higher density in MVM than in BM, while GLUT1 expression in BM increases to approximately 2-fold in the second and the third trimesters, and remains at that level until term ( Illsley and Baumann, 2020 Illsley and Baumann, 2020 ). The expression of GLUT3 is primarily in MVM, and it decreases significantly with increasing gestational age ( Brown et al., 2011 Brown et al., 2011 ). GLUT4 is expressed in the cytosol of syncytial cells in the first trimester, which can be recruited rapidly to the cell surface under insulin stimulation. The expression of GLUT4 significantly decreased in the third trimester ( Illsley and Baumann, 2020 Illsley and Baumann, 2020 ). GLUT4 has long been known as an insulin-responsive GLUT; however, some studies found that its effect on glucose homeostasis can also be independent of insulin. Muscle-specific insulin receptor knockout mice exhibit enlarged fat mass and greatly decreased glucose uptake level when stimulated by insulin, but the muscle glycogen level remains normal ( Brüning et al., 1998 Brüning et al., 1998 ). This indicates a possible insulin-independent pathway for glucose transport, such as adiponectin and leptin ( Xue and Kahn, 2006 Xue and Kahn, 2006 ). Our study revealed that MLT can also significantly increase glucose uptake and GLUT4 protein expression independent of insulin stimulation. Nonetheless, few studies have focused on the effect of MLT on glucose transport under insulin-independent pathways, prompting further research in this field. Yao et al. (2017) Yao et al. (2017) reported that GLUT1 expression is increased in the placenta of pregnant women with GDM, and GLUT1 mRNA and protein expression levels are positively correlated with the birth weight of infants from GDM women with macrosomia. In the placenta of pregestational diabetes and insulin-controlled GDM women, GLUT4 expression was positively correlated with the birth weight of corresponding infants ( Stanirowski et al., 2019 Stanirowski et al., 2019 ). Based on the above studies, we formulated the following hypothesis: the increase in GLUTs in the placenta of GDM leads to the enhanced placental ability of glucose uptake and transport, which creates a high-glucose environment for the offspring, resulting in several adverse fetal outcomes. Therefore, we considered it highly important to explore the related pathways of GLUT expression. Glucose transport is regulated by various signaling pathways. Our study indicated that MLT stimulated glucose uptake and the expression of GLUT4 protein through MT2 receptors in HTR-8/SVneo cells, and PPARγ expression was also increased. PPARγ is a key regulator of glucose uptake and transport, and acts to increase insulin sensitivity and glucose metabolism ( Liao et al., 2007 Liao et al., 2007 ; Montaigne et al., 2021 Montaigne et al., 2021 ). Thus, we assumed that MLT affects the uptake and transport of glucose by modulating the expression of PPARγ, the mechanism of which needs to be elucidated by further research. The present study has some limitations that should be addressed. First, the expression levels of GLUTs were not detected in GDM and NP tissues. Second, although we observed that the effect of MLT on GLUT4 expression can be independent of insulin stimulation, we still need to explore the effect of MLT on GLUT4 in the presence of insulin due to increased insulin secretion in early pregnancy. Third, the regulation of glucose uptake and the expression of GLUTs by PPARγ in trophoblasts requires further investigation. Fourth, the function of MT2 receptor in regulating glucose uptake and transport in vivo is yet to be explored. 4 Conclusions 1 To summarize, this study confirmed that the genetic polymorphism of rs10830963 contributed to high plasma glucose levels (FPG, 1-h and 2-h plasma glucose) and GDM. The MT2 receptor encoded by MTNR1B genes was predominantly expressed by trophoblasts in the placenta and was increased in GDM. In vitro, MLT upregulated glucose uptake and the expression of GLUT4 via MT2 receptor in trophoblasts, which may be mediated by the transcriptional factor PPARγ. These findings suggest that the MTNR1B rs10830963 variant is a potential risk factor for developing GDM. The altered expression of MT2 receptor in the placenta of GDM may be involved in the change of placental glucose uptake and transport levels, which could be associated with abnormal glucose metabolism and macrosomia in children born to GDM mothers. 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J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 23 ( 5 ): 432 - 436 . https://doi.org/10.1631/jzus.B2100753 https://doi.org/10.1631/jzus.B2100753 Prediction of birth weight in pregnancy with gestational diabetes mellitus using an artificial neural network Zhu B , Ma ZX , Zhu YN , et al. , 2021 . Reduced glycodeoxycholic acid levels are associated with negative clinical outcomes of gestational diabetes mellitus . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 3 ): 223 - 232 . https://doi.org/10.1631/jzus.B2000483 https://doi.org/10.1631/jzus.B2000483 Reduced glycodeoxycholic acid levels are associated with negative clinical outcomes of gestational diabetes mellitus Lijie WEI performed the experiments and prepared this manuscript. Peng GAO, Jingyi ZHANG, and Xuan ZHOU were in charge of obtaining written consent from participants, collecting clinical and biochemical data, and confirming the data accuracy. Huiting ZHANG, Yi JIANG, and Yuanyuan DU participated in experimenting. Xuan GAO, Yuting CHEN, and Jiaqi LI were responsible for data analysis and results discussion. Shenglan ZHU, Chenyun FANG, and Mengzhou HE proofread and adjusted the manuscript. Shaoshuai WANG, Ling FENG, and Jun YU conceptualized and provided methodology opinions. All authors have read and approved the final manuscript, and therefore, they have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the Health Commission of Hubei Province Scientific Research Project (No. WJ2021M129) and the National Key Research and Development Program of China (No. 2021YFC2701502). 10.1631/jzus.B2200136 1673-1581（2023）01-0078-11 韦丽杰,江一,高鹏等.褪黑素受体1B基因多态性及其对葡萄糖转运的调控在妊娠期糖尿病中的作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(01):78-88. 韦丽杰,江一,高鹏等.褪黑素受体1B基因多态性及其对葡萄糖转运的调控在妊娠期糖尿病中的作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(01):78-88. This work was supported by the Health Commission of Hubei Province Scientific Research Project (No. WJ2021M129) and the National Key Research and Development Program of China (No. 2021YFC2701502). 2022-03-19 2022-08-05 2023-01-15 2023-02-06 2022-12-16 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 Lijie WEI, Yi JIANG, Peng GAO, Jingyi ZHANG, Xuan ZHOU, Shenglan ZHU, Yuting CHEN, Huiting ZHANG, Yuanyuan DU, Chenyun FANG, Jiaqi LI, Xuan GAO, Mengzhou HE, Shaoshuai WANG, Ling FENG, and Jun YU declare that they have no conflict of interest. This study protocol was approved by the Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology Ethics Committee (No. TJ-IRB20170506), and was performed under the Declaration of Obstetrics Department. Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. Compliance with ethics guidelines Supplementary information 1 Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 1 24