Gestational diabetes mellitus (GDM) is a metabolic disease associated with insulin resistance. It is characterized by glucose intolerance with first onset during pregnancy. This disease is closely related to obesity and chronic inflammation in adipose tissue.1 Excess adiposity and adipocyte dysfunction lead to dysregulation of a large number of adipose tissue-derived secretory factors, named adipokines, which may result in the development of various metabolic diseases.2,3 Recent studies have found the role of various adipokines in gestational diabetes in order to elaborate the potential relationship between adipokines and gestational diabetes.4
In the past decade, a secreted chemoattractant protein, referred to as chemerin, was identified as a novel adipokine associated with obesity and metabolic syndrome, which regulates adipocyte differentiation and metabolism and is closely related to insulin resistance.5 It has been reported that chemerin could induce insulin resistance in primary human skeletal muscle cells.6 Recent literature reviews7,8 pointed out that the current findings of how chemerin was associated with obesity and insulin resistance were inconsistent. The study of chemerin is still in its infancy and more work needs to be done to elucidate the mechanism of chemerin related to obesity and its metabolic complications.
Different cell lines have been applied to investigate trophoblast development and physiology. However, most cell lines are established from choriocarcinoma, while HTR-8/SVneo is isolated from normal first trimester placenta and transfected with a plasmid including the simian virus 40 large T antigen.9 HTR-8/SVneo is considered as a more similar model of trophoblast cell, because the HTR-8/SVneo cell lines were created by immortalizing the physiologic extravillous trophoblast cell.10
Previous studies have showed that different dose and duration of chemerin treatment contributed to unequal outcomes of insulin stimulated glucose uptake.11 It is tempting to speculate that the effect of chemerin follows a dose-dependent response. Since no information was available on the effect of different density of chemerin on the phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B (PKB) pathway and the mitogen-activated protein kinase (MAPK) pathway in the first trimester trophoblast cells, the present study examined the effects of chemerin levels at 20 ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL, and 100 ng/mL on insulin signaling pathways in high glucose treated HTR-8/SVneo cells.
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor superfamily of ligand-dependent transcription factors, including three isotypes α, δ, and γ. All of them were critical in the storage and mobilization of lipids, in glucose metabolism, in morphogenesis and inflammatory response.12 Among them, PPARγ is of special interest because it can be activated by thiazolidinedione (TZD) class of antidiabetic drugs (potent insulin sensitizers), such as rosiglitazone and pioglitazone.13 PPARγ activation downgraded resistin gene so as to improve insulin resistance.14
Given rosiglitazone's ability to promote insulin sensitivity, we hypothesized that PPARγ activation could regulate chemerin and lead to the alteration of insulin signaling pathway in high glucose treated HTR-8/SVneo cells.
Materials and methods
Cell culture and treatment
HTR-8/SVneo cells were obtained from the Wuhan Servicebio Technology Co., Ltd, China and were cultured in Dulbecco's modified eagle medium (DMEM, Gibco, Thermo Fisher Scientific, Logan, Utah, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. To mimic maternal diabetic circulatory glucose levels in GDM,15,16 Dulbecco's modified eagle medium enriched with glucose at the rate of 4.5 g/L which corresponds to 25 mmol/L of glucose (1 mol glucose = 180.16 g/L) was used. Recombinant human chemerin (#300-66, PeproTech Inc., Rocky Hill, New Jersey, USA), PPARγ agonist rosiglitazone (HY-17386, MedChem Express, Princeton, New Jersey, USA), agonist GW1929 (HY-15655, MedChem Express, Princeton, New Jersey, USA) and PPARγ inhibitor GW9662 (HY-16578, MedChem Express, Princeton, New Jersey, USA) were additionally added to the medium, respectively. This study had approval by a decision of the Ethics Committee of Tongji Medical College, Wuhan, China (decision number: TJ-IRB20170506).
Small interfering RNA (si-RNA) transfection
Transient transfection of si-RNA of chemokine-like receptor 1 (si-CMKLR1, target sequence TGGTCAATGCTCTAAGTGA, Guangzhou RiboBio Co., Ltd, China) and si-RNA of G protein-coupled receptor 1 (si-GPR1, target sequence TGTGGAGTTCAATAATCAT, Guangzhou RiboBio Co., Ltd, China) in HTR-8/SVneo were established using Lipofectamine 3 000 transfection reagent (Invitrogen, Carlsbad, California, USA).
HTR-8/SVneo cells were treated by 0.5%Triton X-100 and after blocking with 5% bovine serum albumin (BSA, G5001-100, Wuhan Servicebio Technology Co., Ltd, China) at room temperature for 2 hours, they were incubated with the following antibodies: anti-chemerin mouse polyclonal antibody (ab72965, Abcam Plc., UK, 5 μg/mL), and Alexa Fluor 594 donkey anti-mouse IgG (H + L) (ANT029, Wuhan Antgene Biotechnology Co., Ltd., China, 1:200). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, G1012, Wuhan Servicebio Technology Co., Ltd, China), and sealed via anti-fluorescence quencher. Observing the image by fluorescence microscope (Olympus, Japan).
Western immunoblot analysis
Cell sediments after treating for 72 hours were homogenized in radio immunoprecipitation assay buffer (P0013B, Beyotime Biotechnology, China) and phenylmethanesulfonyl fluoride (ST506, Beyotime Biotechnology, China) was added with a 1:100 ratio. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P Transfer Membranes, Millipore Sigma, St. Louis, Missouri, USA). After blocking with 5% BSA for 1 hour in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature, the membranes were incubated overnight at 4°C with appropriate dilution of antibodies: anti-chemerin rabbit polyclonal antibody (10216–1-AP, Proteintech, China, 1:200), anti-PPARγ rabbit monoclonal antibody (ab191407, Abcam Plc., UK, 1:1 000), anti-GPR1 rabbit monoclonal antibody (ab157209, Abcam Plc., UK, 1:1 000), anti-CMKLR1 rabbit polyclonal antibody (ab64881, Abcam Plc., UK, 1:500), anti-PI3K p110β rabbit polyclonal antibody (21739–1-AP, Proteintech, China, 1:500), anti-AKT2 rabbit monoclonal antibody (#3063, Cell Signaling Technology, Inc., Boston, Massachusetts, USA, 1:1 000), anti-Phospho-AKT2 rabbit polyclonal antibody (A7006, Assay Biotechnology Company, Inc., Los Angeles, California, USA, 1:500), anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, Cell Signaling Technology, Inc., USA, 1:1 000), anti-Phospho-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4370, Cell Signaling Technology, Inc., USA, 1:2 000), anti-NF-κB p65 rabbit polyclonal antibody (10745–1-AP, Proteintech, China, 1:1 000), anti-β-actin mouse monoclonal antibody (66009-1-Ig, Proteintech, China, 1:5 000). Then the membranes were washed with TBST five times, 5 minutes each time, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (diluted 1: 3 000 in 5% BSA) for 60 minutes. β-actin was used as a control for lane loading. Immunoblots were developed using ECL chemiluminescence kit (G2014, Wuhan Service bio Technology Co., Ltd, China) and visualized by G box chemi XRQ (Syngene, UK). Densitometric analyses were performed using GeneTools from Syngene.
Real time quantitative-polymerase chain reaction (PCR)
The TRIzol regent (#15596018, Invitrogen Life Technologies, USA) was applied to extract RNA from cell sediments after treating for 48 hours, and equal amounts of RNA were reverse transcribed. The purity of the extracted RNA was tested photometrically, and the optimal range of the optical density at 260/280 nm was 1.8–2.0. The complementary DNA were then amplified according to reverse kit (FSQ-101, TOYOBO, Japan) instructions. Reaction includes 1 μg RNA, 2 μL 5× reverse transcription buffer, 0.5 μL reverse transcription enzyme mix, 0.5 μL primer mix, and nuclease-free water was added to a total volume of 10 μL. They were incubated at 65°C for 5 minutes, and then instantly cooled on ice, followed by placing at 37°C for 15 minutes, and 98°C for 5 minutes and stored at -20°C. Real time quantitative- PCR primers were:
- human PI3K forward 5′-GAAGCACCTGAATAGGCAAGTCG-3′;
- human PI3K reverse 5′-GAGCATCCATGAAATCTGGTCGC-3′;
- human AKT2 forward 5′-CATCCTCATGGAAGAGATCCGC-3′;
- human AKT2 reverse 5′-GAGGAAGAACCTGTGCTCCATG-3′;
- human MAPK1 forward 5′-ACACCAACCTCTCGTACATCGG-3′;
- human MAPK1 reverse 5’-TGGCAGTAGGTCTGGTGCTCAA-3′;
- human β-actin forward 5′-CCTTCCTGGGCATGGAGTC-3’;
- human β-actin reverse 5′-TGATCTTCATTGTGCTGGGTG-3′.
Real time quantitative-PCR was performed in a final volume of 20 μL reaction, containing 2 μL template complementary DNA, 10 μL SYBR® green realtime PCR master mix (QPK-201, TOYOBO, Japan), 0.8 μL PCR forward primer (10 μmol/L), 0.8 μL PCR reverse primer (10 μmol/L) and 6.4 μL dH2O (sterile distilled water). The PCR was implemented according to the following parameters: 95°C for 30 seconds, 40 cycles at 95°C for 5 seconds, 55°C for 10 seconds, 72°C for 15 seconds, and ended at a melting curve analysis.
Independent sample t test was performed and the results were expressed as the mean ± standard deviation of three independent experiments. All data were analyzed using the Prism software 5.0 (GraphPad, Inc, San Diego, CA, USA) and were considered significant at P < 0.05.
Chemerin existed in the high glucose treated HTR-8/SVneo cells
To determine whether chemerin was expressed in HTR-8/SVneo cells, which were exposed to 25 mmol/L of glucose, immunofluorescence assay was carried out. The image showed that chemerin was expressed in the cell membrane and cytoplasm (Fig. 1A).
Effects of chemerin on PI3K-AKT pathway and MAPK (ERK1/2) pathway were dependent on concentrations of chemerin
In order to investigate the relationship between chemerin and insulin signaling pathways, that is PI3K-AKT and ERK1/2, different doses of additional recombinant human chemerin were set in high glucose treated HTR-8/SVneo cells.HTR-8/SVneo cells were pre-incubated with 25 mmol/L of glucose for several generations, and then a concentration gradient of chemerin (20 ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL, and 100 ng/mL) were added to the medium. Harvesting whole cell lysates for western blotting after incubating with chemerin for 72 hours was done. The results showed that only ERK2 was expressed in the HTR-8/SVneo cells (Fig. 2A) and 100 ng/mL of chemerin increased the protein expression of PI3K p110β and p-ERK2 (Figs. 2B, 2D) compared to control group (the group without additional chemerin). The protein expression of p-AKT2 remained unaffected when the cells were challenged with 100 ng/mL of chemerin (Fig. 2C). When the concentration of chemerin was less than 100 ng/mL, the distinct effects emerged. Chemerin at 20 ng/mL and 60 ng/mL also enhanced the protein expression of PI3K p110β (Fig. 2B). Relative expression of p-AKT2 was upregulated significantly by 20 ng/mL and 80 ng/mL of chemerin (Fig. 2C) and the relative protein expression of p-ERK2 was also elevated by 60 ng/mL of chemerin (Fig. 2D). Considering that chemerin is a proinflammatory cytokines, NF-κB p65 was applied to verify its effect too. The protein expression of NF-κB p65 was obviously improved by chemerin at 100 ng/mL (Fig. 2E).
Effects of PPARγ on the mRNA expressions of PI3K, AKT2 and MAPK1
Since the activation of PPARγ, such as rosiglitazone, is known as a potent insulin sensitizer, we selected rosiglitazone and another kind of PPARγ agonist GW1929 to investigate the effect of PPARγ on the expressions of PI3K, AKT and MAPK1/ERK2 genes. A PPARγ antagonist GW9662 was also included. Cells were cultured with 25 mmol/L of glucose and we collected cell sediments for real time quantitative-PCR after incubating with rosiglitazone (10 μmol/L), GW1929 (10 μmol/L), and GW9662 (5 μmol/L) for 48 hours, respectively. The results showed that rosiglitazone increased the mRNA levels of PI3K and AKT2 (Figs. 3A, 3B). Further, GW1929 caused the improvement of PI3K and MAPK1 genes (Figs. 3A, 3C), while treating cells with GW9662 resulted the opposite effects. GW9662 significantly decreased the mRNA expressions of AKT2 and MAPK1 (Figs. 3B, 3C).
Effects of PPARγ on chemerin and its receptors in HTR-8/SVneo cells
To examine whether the activation of PPARγ influences the protein expressions of chemerin and its receptors, that is CMKLR1 and GPR1, HTR-8/SVneo cells treated with 25 mmol/L of glucose were incubated with rosiglitazone (10 μmol/L)and GW1929 (10 μmol/L) for 72 hours, respectively. Cell lysates for western blotting were harvested. Our findings showed that rosiglitazone upregulated the protein level of GPR1 concurrently with elevated PPARγ activity (Figs. 4A–C). The relative expressions of PPARγ, chemerin, and CMKLR1 were significantly reinforced simultaneously by GW1929, compared with control group (Figs. 4A–C).
Receptors of chemerin played a key role in PPARγ mediating PI3K-AKT pathway and MAPK (ERK1/2) pathway
To identify the role of chemerin in the process of PPARγ enhancing PI3K-AKT pathway and MAPK (ERK1/2) pathway, we transfected small interfering RNA (siRNA) into HTR-8/SVneo cells to silence two receptors of chemerin. There were six groups in this test: blank (cells without any treatment), negative control (NC, scrambled siRNA transfected cells), siRNA (si-CMKLR1 or si-GPR1 transfected cells), siRNA + rosiglitazone, siRNA + GW1929, siRNA + GW9662. The last three groups indicated that HTR-8/SVneo cells were first transfected by si-CMKLR1 or si-GPR1, and after transfection for 6 hours, rosiglitazone, GW1929 and GW9662 were added to the cell medium, respectively. After 72 hours, cells were lysed to measure the protein expressions of PI3K p110β, AKT2, and ERK1/2 by western blotting analysis. The expressions of CMKLR1 and GPR1 were also revealed (Figs. 5A, 6A). As shown in Figs. 5D, 6D, compared to the NC group, CMKLR1 and GPR1 were silenced well. The protein level of PI3K p110β was downgraded in si-CMKLR1 and si-GPR1 group compared to the NC group (Figs. 5B, 6B). However, incubation with rosiglitazone and GW1929 did not exert distinguished effect on the expression the PI3K p110β on the basis of si-CMKLR1 (Fig. 5B). Rosiglitazone and GW1929 obviously improved the protein expressions of total-AKT2 and phospho-ERK2 (Figs. 5C, 5E), while the expression of phospho-AKT2 remained unaffected without CMKLR1 (P > 0.05, data not shown). In contrast, rosiglitazone and GW1929 still elevated distinctly the expressions of PI3K p110β and total-AKT2 after silencing GPR1 (Figs. 6B, 6C), whereas, rosiglitazone and GW1929 were unable to enhance the expression of phospho-ERK2 in the absence of GPR1 (Fig. 6E). Unfortunately, the expression of phospho-AKT2 remained uncertain in our experiment after silencing GPR1. All in all, we concluded that CMKLR1 had an impact on PPARγ mediating PI3K-AKT pathway and GPR1 was crucial in PPARγ mediating MAPK (ERK1/2) pathway.
GDM, one of the common complications of pregnancy, is associated with impaired β-cell function and decreased insulin sensitivity.17 As is commonly known, GDM is closely related to insulin resistance. The effect of insulin on tyrosine phosphorylation of the insulin receptor was significantly lower in GDM when compared with normal pregnancy.18 In GDM pregnancies, hyperglycemia and enlarged adipose tissue mass could enhance inflammatory responses from leukocytes and endothelial cells, increasing systemic inflammation.19 It has been reviewed that the pathological adipose tissue during pregnancy secretes numerous pro-inflammatory adipocytokines, such as leptin, resistin and tumor necrosis factor-α (TNF-α),3 thus, circulating concentrations of pro-inflammatory cytokines are increased in GDM.4 The role of adipokines in GDM pregnancies has been reported.20–22 Chemerin, encoded by the gene retinoic acid receptor responder 2 (RARRES2), is a novel adipocytokine associated with obesity and metabolic disease.23 Chemerin is mostly expressed and secreted in adipocytes, but it has been confirmed that it also exists in other tissues including placenta, ovary, and liver.5 Chemerin is a pro-inflammatory chemokine when it was first discovered, promoting the inflammation and dysfunction of adipose tissues in obesity.24 Findings in the last decade have indicated a multifaceted role for chemerin, including inflammation, adipogenesis, angiogenesis and energy metabolism.7
There are two main insulin signaling pathways: the PI3K-AKT/PKB pathway and the MAPK (ERK1/2) pathway. Activation of PI3K and AKT plays a critical role in metabolic regulation.25 PI3K family has three of the class I catalytic isoforms: p110α, β and δ, and they are collectively known as the class IA subgroup.26 Among them, the p110β isoform is a coincidence site for GPCR and tyrosine kinase signaling.27 PI3K mediates activation of AKT2, which promotes GLUT4 translocation and glucose uptake into the cell.17 Mice deficient in AKT2 developed type 2 diabetes mellitus.28 The MAPK pathways are not involved in metabolic actions of insulin but rather in mediating the effect of insulin on mitogenesis and cellular growth.3 ERK1 and ERK2 are members of the MAPK family. Recombinant chemerin induced the phosphorylation of ERK1/2 in differentiated 3T3-L1 adipocytes.29 ERK2 is dispensable for mesoderm differentiation.30 ERK1 is not expressed in early-stage embryos, but ERK2 is required for mouse trophoblast development and has a specific function in the developing embryo.31 Our results indicated that only ERK2, also called MAPK1, was expressed in HTR-8/SVneo cells (a first trimester trophoblastic cell line), corresponding to previous evidence.
It has been widely demonstrated that the functions of chemerin vary depending on cell type. According to previous studies, chemerin and CMKLR1 were expressed in mature 3T3-Ll adipocytes5 and might play a role of enhancing insulin signaling in it.32 During the differentiation of 3T3-L1 cells, the mRNA expressions of chemerin and its receptors were significantly increased.29 In addition, both human species and mouse have an expression of CMKLR1 in dendritic cells and macrophages.33,34 Furthermore, chemerin exerted anti-inflammatory effects on activated macrophages.35 In human microvascular endothelial cells, chemerin was shown to activate MAPK p38, ERK, and AKT.36 However, some scholars have discovered chemerin induced insulin resistance in primary human skeletal muscle cells.6 In the present study, we used high glucose (25 mmol/L) treated HTR-8/SVneo cells to mimic the glucose level of fetal-maternal interface in GDM placenta, we noticed that chemerin was expressed in HTR-8/SVneo cells and recombinant human chemerin (100 ng/mL) in vitro activated the protein expression of PI3K p110β, phospho-ERK2, and NF-κB p65. Consistently, when incubated with 100 ng/mL recombinant human chemerin, insulin-stimulated glucose uptake was improved by 41%.32
Different density and length of time of additional chemerin led to different results. At low picomolar concentrations, chemerin exerted anti-inflammatory effects in vitro.35 Pretreatment with chemerin (1–300 ng/mL, 24 hours) dramatically inhibited phosphorylation of NF-κB to exert anti-inflammatory roles in human vascular endothelial cells.37 Another study which implicated that chemerin might be a protective adipocytokine manifested that the optimal concentration and reaction time of chemerin were 500 ng/mL and 24 hours.38 In human skeletal muscle cells, chemerin was revealed to activate MAPK p38, NF-κB, and ERK, while administration with 1 μg/mL of chemerin significantly decreased phosphorylation of AKT.6 Of particular significance in our experiments was the demonstration that adding chemerin for 72 hours exerted contradictory impacts on PI3K p110β, p-ERK2, p-AKT2, and NF-κB p65 when the concentrations were less than 100 ng/mL. The dose-dependent effects were mirrored by chemerin.
The TZDs, one group of PPARγ agonists,13 ameliorated insulin resistance by regulating TNF-α and free fatty acids.39 Our data also provided the evidence that rosiglitazone, a well known type of TZDs, induced the elevated mRNA levels of PI3K and AKT2 in HTR-8/SVneo cells. In addition, GW1929 (PPARγ agonist) activated the mRNA expressions of PI3K and MAPK1. Conversely, GW9662 (PPARγ antagonist) downgraded the mRNA expressions of AKT2 and MAPK1. PPARγ plays a dominant role in adipose cell differentiation40 and is generally known as the leading regulator of adipogenesis. The increased expression of PPARγ in adipogenesis immediately induces the expression of GLUT4 and the insulin receptor.41 Adipose-specific knock-out of PPARγ was found to cause a severe decrease in adipocyte function, contributing to insulin resistance, and many other metabolic disturbance.42,43
There are three human chemerin receptors: chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1) and chemokine (C-C motif) receptor-like 2 (CCRL2), but the binding of chemerin to CCRL2 does not induce downstream signaling pathways.7 Chemerin binds to both CMKLR1 and GPR1 with similar affinity, but with lower affinity to CCRL2.44 CMKLR1 and GPR1 are both members of GPCRs. The International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification recommends that the official name of CMKLR1 is chemerin receptor 1 and GPR1 is chemerin receptor 2.45 Similar to CMKLR1, GPR1 was also involved in adipogenesis and glucose homeostasis.46–48
It has been demonstrated that the up-regulation of CMKLR1 mRNA by TZDs treatment might be due to the activation of C/EBP-α,29 which exists in the promoter region of human CMKLR1 gene.49 Another sequence analysis exposed that an assumed PPARγ response element sequence exists in the chemerin promoter.50 Similarly, our findings revealed that in addition to significantly up-regulating the protein expression of PPARγ, rosiglitazone raised protein level of GPR1. At the same time, GW1929 upgraded the protein levels of chemerin and CMKLR1. To investigate whether rosiglitazone and GW1929 enhances insulin signaling pathways via chemerin, we silenced the two main chemerin receptors: CMKLR1 and GPR1. However, when CMKLR1 was silenced, rosiglitazone and GW1929 exerted no effect on the expression of PI3K p110β and phospho-AKT2. Meanwhile, the expression of phospho-ERK2 remained unaffected in the absence of GPR1. These results illustrated that CMKLR1 had an effect on PPARγ mediating PI3K-AKT pathway and GPR1 played a crucial role in PPARγ inducing MAPK (ERK1/2) pathway.
In summary, our results revealed a potential mechanism of action of chemerin in insulin signaling pathways and established that an important function of PPARγ was to regulate the expressions of chemerin and its receptors. Given our findings in high glucose treated HTR-8/SVneo cells in vitro, it is tempting to assume that both rosiglitazone and GW1929 play a vital role in improving insulin sensitivity via inducing the expression of chemerin. However, there were some shortcomings in our study. The question whether high glucose treated HTR-8/SVneo cells in vitro are similar to maternal circulatory glucose level in GDM remains unknown. Further research will help to clarify the significance of PPARγ regulating chemerin expression in GDM.
This study was supported by the National Key R & D Program of China (No. 2016YFC1000405 and No. 2018YFC1002903).
Xuan Zhou designed the research, performed the experiments, drafted the manuscript; Li-Jie Wei and Jia-Qi Li conducted the statistical analysis. All authors discussed the results and revised the manuscript.
Conflicts of Interest
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