Glucose uptake in trophoblasts of GDM mice is regulated by the AMPK-CLUT3 signaling pathway

Glucose uptake in trophoblasts of GDM mice is regulated by the AMPK-CLUT3 signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Glucose uptake in trophoblasts of GDM mice is regulated by the AMPK-CLUT3 signaling pathway Zhenghua Xiao, Xue liu, Xiaojin Luan, Ran Duan, Wei Peng, Chao Tong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3680631/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Introduction : GDM as a metabolic disease during pregnancy, regulates GLUT3 translocation by AMPK, thereby affecting glucose uptake in trophoblasts. It provides a new research idea and therapeutic target for alleviating intrauterine hyperglycemia in GDM. Methods : STZ was used to construct GDM mice, inject AICAR into pregnant mice, and observe fetal and placental weight; flow cytometry was employed for the detection of glucose uptake by primary trophoblast cells; immunofluorescence was applied to detect the localization of GLUT3 and AMPK in placental tissue; Cocofal microscope was used to detect the localization of GLUT3 in trophoblast cells;qRT-PCR and Western blot experiments were carried out to detect the expression levels of GLUT3 and AMPK in placental tissue; CO-IP was utilized to detect the interaction of GLUT3 and AMPK. Results : Compared with the normal pregnancy group, the weight of the fetus and placenta of GDM mice increased (P<0.001), and the ability of trophoblasts to take up glucose decreased (P<0.001).In addition, AMPK activity in trophoblasts and membrane localization of GLUT3 in GDM mice were down-regulated compared with normal pregnant mice (P<0.05). There is an interaction between GLUT3 and AMPK. Activating AMPK in trophoblasts can up-regulate the expression of GLUT3 membrane protein in trophoblasts of mice (P<0.05) and increase the glucose uptake of trophoblasts (P<0.05). Discussion : Inhibition of AMPK activity in GDM mice results in aberrant localization of GLUT3, which in turn attenuates glucose uptake by placental trophoblast cells.AICAR activates AMPK to increase the membrane localization of GLUT3 and improve the glucose uptake capacity of trophoblasts. Gestational diabetes mellitus GLUT3 AMPK Glucose uptake AICAR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Gestational diabetes mellitus(GDM) is one of the most common complications during pregnancy, affecting about 8–25% of pregnancies [ 1 ] . The overall prevalence of GDM in mainland China is about 14.8%, and it has continued to increase in the past 10 years [ 2 ] .GDM refers to a condition in which women without diabetes develop diabetes during pregnancy, which usually occurs in the middle or late pregnancy, and is related to insulin resistance, hyperglycemia, and maternal hyperinsulinemia [ 3 – 4 ] .GDM may have an adverse effect on the health of mothers and offspring. Women with GDM are more likely to cause pregnancy complications such as miscarriage, dystocia, preeclampsia, fetal malformations, perinatal macrosomia, and neonatal hypoglycemia [ 5 – 6 ] . In addition, GDM may also increase the risk of type 2 diabetes, cardiovascular disease, childhood obesity, etc [ 7 – 8 ] . Unfortunately, the pathogenesis of GDM remains unclear, leading to a lack of targeted treatment for its underlying cause. Glucose, as the main energy substrate, is essential for the growth and development of the fetus and placenta. During pregnancy, the glucose transporters(GLUTs) transport glucose from the maternal circulation to the fetus by facilitated diffusion in the placenta [ 9 – 10 ] . The GLUTs family includes 14 subtypes, which transport glucose and other nutrients across different tissues [ 11 ] . Studies have shown that GLUT-1, GLUT-3, GLUT-4, GLUT-8, and GLUT-12 have been detected in human placental tissues, among which GLUT1, GLUT3, and GLUT4 have been reported as the main glucose transporters [ 12 – 14 ] .GLUT4 as an insulin-sensitive glucose transporter, is mainly distributed in the surrounding tissues such as fat, myocardium, and skeletal muscle, and is regulated by the insulin signaling pathway [ 15 – 16 ] . Studies have found that the expression of GLUT4 is reduced in the placenta tissue of GDM women [ 17 ] . The glucose metabolism of trophoblasts is possible to be regulated through the IRS1/GLUT4 signaling pathway in mice fed with a high-fat diet after FGF is given [ 18 ] . At present, the uptake and transport of glucose by the placenta are mainly mediated by GLUT1 and GLUT3 [ 19 ] . GLUT1 is ubiquitously expressed in placental tissues and is responsible for basic glucose transport [ 20 ] . The expression pattern of GLUT3 is limited, only in placental trophoblasts, and GLUT3 has a high affinity for glucose, while the affinity for other monosaccharides (such as mannose, xylose, and galactose) is much lower, indicating that GLUT3 plays a special role in the process of glucose transport in the placenta [ 21 – 22 ] . Studies have shown that in the mouse model of Intrauterine Growth Restriction(IUGR), the expression level of placental GLUT3 is down-regulated, and the glucose transported through the placenta is also reduced [ 23 ] . The above proofs indicate that the changes of GLUT3 in placental tissue may play an important role in glucose uptake and metabolism. Adenosine 5‘-monophosphate-activated protein kinase(AMPK) acts as an important energy sensor, which can be activated to promote decomposition metabolism and reduce synthetic metabolism. AMPK is a relatively complex heterotrimeric serine/threonine kinase, consisting of a catalytic (A1, A2) subunit and two regulation (B1, B2, and γ1, gamma 2 or γ3) subunits, Get active by A2 subunit 172-site threonine phosphorylation [ 24 ] . AMPK controls the energy metabolism and glucose steady state of skeletal muscle, liver, adipose tissue, and pancreatic β cells. The AMPK pathway is the major target in preventing many metabolic diseases, including cancer, fatty liver diseases, and diabetes [ 25 – 27 ] . Evidence suggests that activation of AMPK in skeletal muscle cells can promote GLUT4 film transition, consequently increasing glucose intake [ 28 ] . However, a few studies focused on the relationship between AMPK and GLUT3 have found that AMPK in the activation of colon cancer cells can promote GLUT3 expression, and the level of GLUT3 is significantly up-regulated in the use of the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) [ 29 ] . However, it remains unclear whether the AMPK-GLUT3 regulatory axis exists in the mouse placenta. Therefore, in this study, an animal model of GDM was constructed and AICAR was used to stimulate pregnant mice to study the possible role of AMPK-GLUT3 in mouse placental tissue. Materials and methods Chemicals and assay kits BCA Protein Assay Kit (catalogue #: P0011), Membrane and Cytosol Protein Extraction Kit (catalogue #: P0033), Trypsin-EDTA Solution (catalogue #: C0201), BeyoColor™ Prestained Color Protein Marker (catalogue #: P0076), SDS-PAGE Sample Loading Buffer (catalogue #: P0015L), BeyoECL Moon (catalogue #: P0018FS)were purchased from Beyotime (Shanghai, China). Phospho-AMPKa (Thr172) (40H9) Rabbit mAb(catalogue#:2535) and AMPKa Antibody(catalogue #:2532)were acquired from CST (Boston,USA). Streptozocin(catalogue #S0130), PVDF membranes (catalogue #: SAMPLG001), Protein G–Agarose ༈catalogue #P7700), Dulbecco’s phosphate-buffered saline (DPBS) (catalogue #: 524650), reduced-serum medium (DMEM) (catalogue #: M3817), foetal bovine serum (FBS) (catalogue #: 12103C) and Collagenase (catalogue #: C5138)were obtained from Sigma (Japan). Percoll(catalogue #: P8370), Sodium Citrate buffer(catalogue #: C1013) and Proteinase K(catalogue #: 39450-01-6) were purchased from Solarbo (beijing,China). Glut3 Antibody(catalogue #: sc-74399) was procured from SCB (Japan). D-Glucose and 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)- 186689-07-6(catalogue #: N13195)were bought from ThermoFisher (USA). AICAR༈catalogue #: S1802༉ was acquired from Selleck.cn (Shanghai, China). HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L)(catalogue #:SA00001-1), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L)(catalogue #:SA00001-2), Fluorescein (FITC)–conjugated Affinipure Goat Anti-Rabbit IgG(H + L)(catalogue #:SA00003-2), R-PE-conjugated Goat Anti-Rabbit IgG(H + L)(catalogue #:SA00008-2), Cytokeratin 7-Specific Polyclonal Antibody(catalogue #:17513-1-AP), AMPK Alpha Monoclonal Antibody(catalogue #:66536-1-Ig) and Alpha Tubulin Polyclonal Antibody(catalogue #:11224-1-AP) were purchased from Proteintech(USA). Acryl/Bis 30% Solution(catalogue #: B546016), 1M Tris-HCl Solution, pH 6.8, Sterile(catalogue #: B548122), 4X Tris-HCl/SDS (pH 8.8)(catalogue #: B546021༉, N,N,N’,N’-Tetramethylethylenediamine (catalogue #: A610508), Ammonium persulfate(catalogue #: A100486), 10% SDS Solution(catalogue #: B548118), 10X Tris-Glycine SDS-PAGE Running Buffer(catalogue #: C520001), EZ-Buffers C 10X Western Transfer Buffer(catalogue #: C520003) and EZ-Buffers H 10X TBST Buffer(catalogue #: C520009) were acquired from Sangon Biotech (Shanghai, China). PrimeScript™ RT Master Mix (catalogue #: RR036Q) and TB Green® Premix Ex Taq™(catalogue #: RR420Q) were obtained from Takara (Japan). Animal and experimental design Unfertilized female C57BL/6J mice aged 6–8 weeks, were purchased from Chongqing Tengxin Biological Co.Ltd, and then fed in the SPF animal laboratory of Chongqing Medical University. All procedures were conducted in accordance with ARRIVE guidelines.This study was approved by the Chongqing Medical University Ethics Committee. Mice were raised in the animal laboratory of Chongqing Medical University, on a regular 12-hour night and day cycle, and fed with a standard murine diet and water ad libitum. After being fed to around 9–10 weeks, the female mice were mated with the male mice at a ratio of 1:1. Female mice were examined for the vaginal plug at 8:00 AM the following morning. The day on which a vaginal plug was detected was considered the gestation day 0.5 (GD0.5). The pregnant mice were divided into 4 groups: normal pregnancy group, GDM group, Normal + AICAR group, and GDM + AICAR group, with 3–5 mice in each group. The GDM and GDM + AICAR groups were injected with 150mg/kg of STZ diluted in sodium citrate at GD12.5, while the Normal + AICAR group and GDM + AICAR group were injected with 250 mg/kg AICAR at GD17.5. For the oral glucose tolerance test(OGTT), each group was given 2mg/kg glucose at GD14.5 and GD18.5. Then, blood samples were collected from the tail vein at 0, 30, 60, 90, and 120 minutes to measure blood glucose levels. Tissue collection and preparation All pregnant mice were euthanized at GD18.5, and offspring and placenta were collected. Phosphate-buffered saline (PBS) was pre-cooled to wash the blood of the placenta tissue until the rinse was colorless. Part of the placenta was placed in a cryotube and stored in the refrigerator at -80℃ for qRT-PCR and Western Blot experiments. The tissue sample was embedded in paraffin and stored at room temperature before immunofluorescence experiments. primary trophoblast cells were extracted from the remaining placental for 2-NBDG uptake experiments. 2-NBDG uptake assay First, mouse placental tissue was added to 1 ml of collagenase IV at a concentration of 0.01 g/ml, quickly chopped, and then placed in a 37°C incubator for full digestion of 30 minutes. Then, 5ml of complete medium was added to stop digestion, the cell suspension was sieved with a 70um cell strainer centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded and resuspended in 5ml of stain buffer for later use. Next, percoll mixtures with concentrations of 60%, 40%, 20%, and 5% were prepared and added to a 50ml centrifuge tube in turn. The above cell suspension was slowly added to the uppermost layer. The cell suspension at 20–40% density layer was collected after centrifugation at 1500 rpm for 5 minutes. Next, the mixture was resuspended in 5 ml of complete medium, and the cell pellets were collected after differential adherence for 30 minutes and resuspended and seeded in 24-well plates for 24 hours. The next day, the 24-well plate was washed twice with pre-cooled PBS, and then incubated with 100um of 2-NBDG for 30 minutes. Finally, cells were collected and the intracellular fluorescence intensity was immediately measured by flow cytometry. RNA extraction and quantitative RT-PCR Trizol was used to extract total RNA from the tissue according to the instructions, and the RNA concentration was determined. A 1 mg RNA was reversed transcribed to cDNA using the cDNA reverse transcription kit. The qRT-PCR reaction was performed with SYBRPrem ixExTaqt in the RT-PCR detection system. The gene expression levels were normalized using the housekeeping gene GAPDH. The following is the primer sequence of each gene: GLUT3:Sense5′-GAGATGAAGGATGAGAGTGTTCGGATG-3’, Anti-sense5’-GCTGGAGGACAATGGAGATGAGAAG-3’; AMPK:Sense5′-GCCTTGAAAGAAGTGTGTGAGAAGTTC-3’, Anti-sense5’-GTGGGTCCTGGTGGTTTCTGTTG-3’; GAPDH:Sense5′-ATGTGTCCGTCGTGGATCTGAC-3’, Anti-sense 5′-AGACAACCTGGTCCTCAGTGTAG-3’. Finally, the 2-ΔΔct method was used to calculate the relative expression of sample genes. Protein extraction and western blot 30mg of placental tissue was taken from each sample, RIPA lysate was used for total protein extraction, membrane protein, and cytoplasmic protein extraction refer to kit instructions. The BCA kit was used to determine the protein concentration was determined using the BCA kit. The protein was stored in a refrigerator at -80°C. First, SDS-PAGE was used to separate the proteins, which were then transferred to PVDF membranes. Then, the membrane was blocked in 5% no-fat milk and hybridized overnight at 4℃ with primary antibodies, including AMPK, P-AMPK, GLUT3, and Tubulin. The next day, the membrane was washed 5 times with TBST and incubated with the corresponding secondary antibody for 1 hour. Finally, ECL was used for chemiluminescence detection, and grayscale quantitative analysis was performed by Gel-Proanlyzer software. Immunofluorescence and confocal microscopy Paraffin blocks were sliced, dewaxed, and hydrated. PBS-rinsed sections were incubated with proteinase K for 15 minutes for antigen retrieval. Sections were washed 3 times with PBS for 10 minutes each and then added 0.3% Triton-100 for 30 minutes. Sections were hybridized with primary antibody overnight at 4°C. The primary antibodies include AMPK, GLUT3, CK7, and Na+-K+-ATPase. Next, the sections were washed three times with PBS again, and incubated with the corresponding secondary antibodies for 1 hour. Finally, Hoechst 33442 was added and incubated with the sections for 10 minutes, and Fluorescence microscopy or confocal microscopy was performed to observe the fluorescent signal of the tissue on the slice. Co-Immunoprecipitation For each sample, 30 mg of mouse placental tissue was placed in 1 ml of RIPA lysis buffer and lysed on ice for 30 minutes. Then, 200ul supernatant of the lysate was collected after centrifuging at 16000g at 4°C for 30 minutes, and stored at -80°C. 30ul protein A agarose beads were washed 3 times in 500ul PBS on a shaker. Next, the washed Protein A agarose beads and lysate were mixed with 2ul of AMPK or GLUT3 primary antibody, respectively, and rotated at 4°C overnight. The next day, the mixture was centrifuged at 3000 rpm at 4°C for 3 minutes, the supernatant was discarded, and the pellet was washed 3 times with 500 ul of PBS. Finally, the pellet was resuspended in 30ul of RIPA lysis buffer, and 10ul of loading buffer was added and boiled at 100°C for 10 minutes. The expression levels of AMPK or GLUT3 in each protein sample were detected by western blot. Statistics At least three independent replicates for each experiment. Measurement data are expressed as mean ± SEM). The independent sample t-test was used to compare the two groups, and the one-way analysis of variance was used to compare the multiple groups. P < 0.05 is considered statistically significant. * P < 0.05; ** P < 0.01; *** P < 0.001. Results 1. Decreased glucose uptake capacity of trophoblast cells in GDM mice Pregnant C57 mice were injected intraperitoneally with 150 mg/kg STZ or an equal volume of PBS at GD12.5. OGTT test was performed on two groups of mice at GD14.5. As shown in Fig. 1 A, the blood glucose of the mice in the GDM group was higher than that in the normal group at 0, 30, 60, 90, and 120 minutes after the mice were gavaged with glucose. The area under the curve of blood glucose in the GDM group was also higher than that in the control group (P < 0.001; Fig. 1 .A, B). In addition, the results of the 2-NBDG experiment showed that the glucose uptake capacity of the trophoblasts in the GDM group was significantly decreased (P < 0.001; Fig. 1 .C, D). 2. Offspring and placental weights of GDM mice were increased Euthanize mice at GD18.5 and test the offspring and placental weights of normal pregnancy and GDM mice (Fig. 2 .A). It was found that the weight of the offspring(P < 0.001)and placenta (P < 0.001)of GDM mice were significantly higher than that of the normal pregnancy group(Fig. 2 .B, C). 3. Down-regulation of GLUT3 expression on trophoblast cell membranes in GDM mice We found that CK7 and GLUT3 co-localized in the placental tissue of the normal and GDM groups, and the proportion of GLUT3-positive cells in the GDM group decreased by immunofluorescence (Fig. 3 .A). Confocal microscopy imagine showed that, GLUT3 is expressed in the cell membrane and cytoplasm of both the normal and GDM groups(Fig. 3 .B). Meanwhile, the qRT-PCR experiment showed no significant difference in the transcription level of GLUT3 in the placenta tissue of the two groups (P > 0.05, Fig. 3 . C). Previous studies have shown that GLUT3 exhibits rapid and significant membrane translocation during a marked increase in neuronal glucose uptake. Interestingly, western blot results suggested that there was no difference in the expression levels of total GLUT3 protein and cytoplasmic GLUT3 protein in the placental tissue of the normal and the GDM group (Fig. 3 . D, E, F, G). However, the expression level of GLUT3 protein in the placental tissue of the GDM group was significantly decreased(P < 0.05,Fig. 3 .H, I), which suggested that the level of GLUT3 on the trophoblast cell membranes of GDM mice was down-regulated. 4. p-AMPK was significantly down-regulated in the trophoblast cells of GDM mice As a key competent molecule, activated AMPK plays an important role in glucose uptake. Immunofluorescence showed that CK7 and AMPK also co-localized in the mouse placenta(Fig. 4 . A), and the qRT-PCR results showed no significant difference in the transcription level of AMPK in the placenta between the two groups of mice(Fig. 4 . B). Phosphorylation of AMPK significantly enhances AMPK activity. Therefore, we detected the expression level of p-AMPK in placental tissue by western blot experiment. The results showed no difference in the expression level of AMPK protein in the placenta in the two groups(Fig. 4 .C, D), while the expression level of p-AMPK protein in the placental tissue of mice in the GDM group was significantly decreased(Fig. 4 .E). These results support the significant downregulation of AMPK phosphorylation in trophoblast cells of GDM mice. 5. Interaction between AMPK and GLUT3 in placental tissue The above evidence indicates that GLUT3, AMPK, and CK7 co-localize with each other in mouse placenta tissue. Furthermore, the plasma membrane localization of GLUT3 was significantly down-regulated in the placental tissue of GDM mice, and the phosphorylation level of AMPK was also decreased. Previous studies have also found that activation of AMPK in neurons can promote the membrane translocation of GLUT3. Hence, our study substantiated the interaction between AMPK and GLUT3 in mouse tissue(Fig. 5 ). The findings indicated that AMPK may regulate the translocation of GLUT3 from the cytoplasm to the cell membrane through direct binding. 6. AICAR reduces the blood sugar level of mice and promotes the glucose uptake capacity of trophoblast cells AICAR, a well-known AMPK agonist, has been shown to reduce blood sugar in mice by activating AMPK. To investigate this further, pregnant C57 mice were administered intraperitoneal injections with 250 mg/kg AICAR or an equivalent volume of PBS at GD17.5. Subsequently, an OGTT test was performed on four groups of mice at GD18.5. The results in Fig. 1 A indicated that the blood glucose levels of the mice in the AICAR group were consistently lower than those of the corresponding non-AICAR group at 0, 30, 60, 90, and 120 minutes following glucose administration. Furthermore, the area under the curve for blood glucose was significantly reduced after AICAR injection (P < 0.001; Fig. 6 .A, B). Additionally, the results of the 2-NBDG experiment showed that the glucose uptake capacity of the trophoblast cells also increased after AICAR injection(P < 0.01, Fig. 6 .C, D). 7. AICAR up-regulated the expression levels of p-AMPK and membrane GLUT3 in mouse trophoblast cells Similarly, the qRT-PCR findings indicated no substantial difference in the transcription levels of GLUT3 and AMPK in the placental tissue of the four groups of mice(Fig. 7 .A, B). The western Blot experiments confirmed that AICAR facilitated the phosphorylation of AMPK and the expression of GLUT3 in the mouse trophoblast cell membrane(Fig. 7 .C, D, G, H).In conclusion, AICAR up-regulated the expression of GLUT3 on the trophoblast cell membrane of GDM mice by activating AMPK. Discussion In this study, we used STZ to construct a GDM mice model and found that compared with normal pregnant mice, the expression level of GLUT3 on the trophoblast membrane and the activity of AMPK in the trophoblast of GDM mice were significantly down-regulated.In vivo experiments have confirmed that AICAR can activate AMPK, thereby improving the abnormal localization of mice trophoblasts GLUT3 and increasing the glucose uptake of mice trophoblasts. In general, we speculate that AICAR increases the membrane localization of GLUT3 by upregulating the activity of AMPK in trophoblasts of GDM mice. And enhanced the glucose uptake ability of mouse trophoblast. The transfer of glucose at the interface between the mother and fetus occurs following the concentration gradient of glucose, with high concentrations in maternal blood and low concentrations in the fetus. The intrauterine hyperglycemia environment in GDM expands the glucose concentration gradient between the mother and the fetus, leading to excessive transfer of glucose from the placenta to the fetus. In turn, it affects the secretion of fetal insulin, the growth of fat cells, and the synthesis of triglycerides, ultimately leading to fetal overgrowth [ 30 – 31 ] . Studies have also found that GDM is related to fetal growth retardation and poor growth, An epidemiological survey in the United States shows that about 7% of the offspring of GDM females are small for gestational age(SGA) [ 32 ] .In a retrospective case-control study of 1981 SGA babies conducted in China, 383 SGA babies (19.3%) were born to mothers with GDM [ 33 ] . This study revealed a significant reduction in the glucose uptake capacity of placental trophoblasts in GDM mice compared to those in the normal pregnancy group. Additionally, notable increases was found in both fetal and placental weight in GDM mice. Furthermore, dysplasias were observed in the offspring of GDM mice. These findings suggest an imbalanced glucose metabolism in the placenta of GDM mice, thereby elevating the likelihood of adverse pregnancy outcomes. GLUT3 in mice was first found in brain tissue and showed a high expression pattern [ 34 ] . Studies have found that in the hippocampus of 3XTg-AD mice, the decrease in phosphorylation of AKT in the insulin signaling pathway leads to a decrease in GLUT3 translocation. This reduction in GLUT3 on the cell membrane may lead to impaired neurons' ability to take up glucose [ 35 ] . Existing evidence shows that GLUT3 also plays an important role in the embryonic development of mice and the transfer of glucose from the mother to the fetus, the mouse GLUT3 mutation leads to a decrease in glucose transported through the placenta, resulting in loss of early pregnancy and fetal growth restriction in late pregnancy [ 36 ] . It can be seen that GLUT3 is an important mediator of glucose transport in the placenta [ 37 ] .In this study, we observed that the expression level of GLUT3 on the cell membrane in the placenta tissue of GDM mice was significantly lower than that of the normal pregnancy group, while the total expression level of GLUT3 in the two groups of mice did not change significantly. It shows that GLUT3 has abnormal localization in the placenta tissue of GDM mice. This abnormal localization may be caused by the intrauterine hyperglycemia environment. AMPK regulates the activity of proteins in many important metabolic signaling pathways through the phosphorylation pathway, and is considered to be a key protein related to type 2 diabetes [ 38 ] . It is worth noting that compared with the normal population, the activity of AMPK in the adipose tissue of non-pregnant obese or diabetic individuals was significantly down-regulated [ 39 ] . Although the exact mechanism of GDM is unclear, GDM is the precursor state of type 2 diabetes. GDM women have a 7.34 times higher risk of developing type 2 diabetes after delivery than normal pregnant women [ 40 ] . More and more research focuses on the mechanism of AMPK in GDM. Studies have pointed out that the activation of AMPK can improve inflammation and insulin resistance of skeletal muscle and adipose tissue in women with GDM [ 41 ] . Recent studies have shown that the presence of activated AMPK in the placental tissues of humans and mice is beneficial to the differentiation of the placenta and the growth of the fetus, the expression level of the AMPK gene in the placental tissues of obese women with GDM decreases. [ 42 – 43 ] . Therefore, we speculate that the imbalance of glucose homeostasis in the placenta tissue of GDM mice is related to AMPK. To test this hypothesis, we constructed a GDM mice model. Although the mRNA levels of AMPK in the normal pregnancy group and GDM mice did not change significantly, Western blot experiments showed that compared with the normal pregnancy group, the phosphorylation level of AMPK in the placenta tissue of GDM mice was significantly down-regulated. Immunofluorescence showed that CK7 and AMPK also co-localized in mouse placenta. And through CO-IP detection, there is an interaction between AMPK and GLUT3 in trophoblasts. Therefore, we believe that AMPK in trophoblasts may directly interact with GLUT3 to promote the transfer of GLUT3 in the cytoplasm to the cell membrane, thereby mediating the transport of glucose, and the ingested glucose is metabolized by glycolysis for energy. The AMPK activity is inhibited in the high glucose environment caused by GDM mice, resulting in abnormal localization of GLUT3, which in turn weakens the level of glucose metabolism in the placenta itself, increases the net glucose transport from the maternal circulation to the fetal circulation, eventually affect the growth of the fetal placenta. To further verify the above findings, in this study, in vivo experiments were conducted to investigate whether AMPK activation can regulate the translocation expression of GLUT3 and glucose transport in mice placental tissues.AICAR is a commonly used AMPK agonist,it can be absorbed and metabolized by tissues in the body into ZMP, an analog of AMP, thereby activating AMPK and promoting glucose uptake by tissues [ 44 ] . Studies have found that AICAR stimulation in rats can increase AMPK activity, thereby improving insulin sensitivity and glucose transport in rat muscles [ 45 ] .In our study, The blood glucose levels of pregnant mice at the OGTT 5 time points and the area under the curve injected with AICAR were significantly lower than those of the non-injected group. The glucose uptake capacity of placental trophoblasts is also significantly increased after AICAR injection. It is suggested that AICAR can improve the impaired glucose uptake of placental trophoblasts and promote the glucose metabolism of the placenta itself. We also found that AICAR significantly increased the phosphorylation level of AMPK, and the expression level of GLUT3 on the cell membrane was also significantly increased. Therefore, we concluded that AICAR can activate AMPK, and promote the translocation of GLUT3 from the cytoplasm to the cell membrane in trophoblasts, thereby regulating placental glucose homeostasis. In summary, We have proved that giving AICAR to STZ-induced GDM mice can activate the trophoblasts AMPK, reversing the abnormal localization of GLUT3 in the mice trophoblasts and improving the glucose uptake capacity of the trophoblasts. In-depth discussion of the regulatory mechanism of glucose transport at the maternal-fetal interface, inhibiting excessive glucose transport to the fetus, reducing the adverse effects of the GDM intrauterine hyperglycemia environment on the fetus, and providing new intervention targets for improving the birth outcome of GDM, but this still requires greater Research to illustrate the role of AMPK-GLUT3 signal axis in regulating placental glucose homeostasis. Declarations Data availability statement The data used to conduct this study are available from the corresponding author(Juan Qiao [email protected] ) upon reasonable request. Author contributions JQ ,CTand HQ conceived the study. ZH,XL,XiaoL,RD,and PW performed the experiments and analyzed the data.ZH and XL wrote the manuscript.JQ and HQ edited the manuscript and provided funding for the work. All authors contributed to the article and approved the submitted version. Ethical approval All procedures were conducted in accordance with ARRIVE guidelines.And this study was approved by the Chongqing Medical University Ethics Committee. Funding This work was supported by the National Natural Science Foundation of China（81901508） References Guariguata L., Linnenkamp U., Beagley J., Whiting D R., Cho N H.(2014). Global estimates of the prevalence of hyperglycaemia in pregnancy. Diabetes Res Clin Pract, 103(2), 176-85. doi:10.1016/j.diabres.2013.11.003. Goran, M., Plows, J., & Ventura, E. (2019). Effects of consuming sugars and alternative sweeteners during pregnancy on maternal and child health: Evidence for a secondhand sugar effect.Proceedings of the Nutrition Society,78(3), 262-271.doi:10.1017/S002966511800263X. 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García-Patterson A., Corcoy R., Balsells M., Altirriba O., Adelantado J M., Cabero L., de Leiva A.(1998). In pregnancies with gestational diabetes mellitus and intensive therapy, perinatal outcome is worse in small-for-gestational-age newborns. Am J Obstet Gynecol, 179(2), 481-5. doi:10.1016/s0002-9378(98)70383-7. Chen Juncao., Xiao Huimin., Yang Yong., Tang Yaping., Yang Xiaoqi., Zhang Zhe., Lu Weineng., Yao Jie., Huang Longguang., Liu Xiaoping., Zhou Wei.(2021). Demographic and Clinical Features of Small-for-Gestational-Age Infants Born to Mothers With Gestational Diabetes Mellitus. Front Pediatr, 9(undefined), 741793. doi:10.3389/fped.2021.741793. Simpson Ian A., Dwyer Donard., Malide Daniela., Moley Kelle H., Travis Alexander., Vannucci Susan J.(2008). The facilitative glucose transporter GLUT3: 20 years of distinction. Am J Physiol Endocrinol Metab, 295(2), E242-53. doi:10.1152/ajpendo.90388.2008. 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Curr Opin Cell Biol, 45(undefined), 31-37. doi:10.1016/j.ceb.2017.01.005. Xu X Julia., Gauthier Marie-Soleil., Hess Donald T., Apovian Caroline M., Cacicedo Jose M., Gokce Noyan., Farb Melissa., Valentine Rudy J., Ruderman Neil B.(2012). Insulin sensitive and resistant obesity in humans: AMPK activity, oxidative stress, and depot-specific changes in gene expression in adipose tissue. J Lipid Res, 53(4), 792-801. doi:10.1194/jlr.P022905. Mathieu Irène P., Song Yanna., Jagasia Shubhada M.(2014). Disparities in postpartum follow-up in women with gestational diabetes mellitus. Clin Diabetes, 32(4), 178-82. doi:10.2337/diaclin.32.4.178. Liong Stella., Lappas Martha.(2015). Activation of AMPK improves inflammation and insulin resistance in adipose tissue and skeletal muscle from pregnant women. J Physiol Biochem, 71(4), 703-17. doi:10.1007/s13105-015-0435-7. Carey Erica A K., Albers Renee E., Doliboa Savannah R., Hughes Martha., Wyatt Christopher N., Natale David R C., Brown Thomas L.(2014). AMPK knockdown in placental trophoblast cells results in altered morphology and function. Stem Cells Dev, 23(23), 2921-30. doi:10.1089/scd.2014.0092. Martino J., Sebert S., Segura M T., García-Valdés L., Florido J., Padilla M C., Marcos A., Rueda R., McArdle H J., Budge H., Symonds M E., Campoy C.(2016). Maternal Body Weight and Gestational Diabetes Differentially Influence Placental and Pregnancy Outcomes. J Clin Endocrinol Metab, 101(1), 59-68. doi:10.1210/jc.2015-2590. Choi Ran Hee., McConahay Abigail., Johnson Mackenzie B., Jeong Ha-Won., Koh Ho-Jin.(2019). Adipose tissue-specific knockout of AMPKα1/α2 results in normal AICAR tolerance and glucose metabolism. Biochem Biophys Res Commun, 519(3), 633-638. doi:10.1016/j.bbrc.2019.09.049. Fisher Jonathan S., Gao Jiaping., Han Dong-Ho., Holloszy John O., Nolte Lorraine A.(2002). Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab, 282(1), E18-23. doi:10.1152/ajpendo.2002.282.1.E18. Additional Declarations No competing interests reported. Supplementary Files SupplementaryDatasetFile.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 Mar, 2024 Reviews received at journal 05 Feb, 2024 Reviewers agreed at journal 31 Jan, 2024 Reviews received at journal 26 Jan, 2024 Reviewers agreed at journal 15 Jan, 2024 Reviewers invited by journal 06 Jan, 2024 Editor assigned by journal 02 Jan, 2024 Editor invited by journal 05 Dec, 2023 Submission checks completed at journal 05 Dec, 2023 First submitted to journal 29 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3680631","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":256436107,"identity":"aa01a20b-27d1-4da6-abac-d638876fe0fa","order_by":0,"name":"Zhenghua Xiao","email":"","orcid":"","institution":"Department of Obstetrics, Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenghua","middleName":"","lastName":"Xiao","suffix":""},{"id":256436108,"identity":"917f3ccd-e589-49e7-b6bf-c51d70af970a","order_by":1,"name":"Xue liu","email":"","orcid":"","institution":"Department of Obstetrics, Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"liu","suffix":""},{"id":256436109,"identity":"1b5f4f30-917d-4479-9960-cee9434cb630","order_by":2,"name":"Xiaojin Luan","email":"","orcid":"","institution":"Chongqing Key Laboratory of Maternal and Fetal Medicine, The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaojin","middleName":"","lastName":"Luan","suffix":""},{"id":256436110,"identity":"8b242801-a616-49d3-998b-6c3256cdc9e1","order_by":3,"name":"Ran Duan","email":"","orcid":"","institution":"Department of Obstetrics, The First Affifiliated Hospital of Chongqing Medical 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Qiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYBACNvmHjQ8SKmrkGNsbiNTCx5B82ODBmWPGzD0HiNQix5CWJvmwhTmxfUYCsQ5jOGMgkdjAZsw78/HGGww1NtGEtTD2GBgk7pCRk5ydVmzBcCwtt4GgFmYeg4TEM2zGhrNzzCQYGw4ToYWNx+BAYhtz4v6bZ4jVwsOW2ADS0jiDh1gtEsyHGRKAgczYA/RLAjF+kZ/B2P7zBzgqD2+88aHGhrAWZGAgkUCKcogWUnWMglEwCkbByAAAhsU/nbsTDwUAAAAASUVORK5CYII=","orcid":"","institution":"Department of Obstetrics, The First Affifiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Qiao","suffix":""},{"id":256436114,"identity":"959f42e1-827b-4141-8ff4-fe671ff6d31d","order_by":7,"name":"Hongbo Qi","email":"","orcid":"","institution":"Women and Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Qi","suffix":""}],"badges":[],"createdAt":"2023-11-29 08:59:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3680631/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3680631/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":47738814,"identity":"0f13e48c-9ce5-42cd-94f3-5a343c7d05f8","added_by":"auto","created_at":"2023-12-06 18:54:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":201467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased glucose uptake capacity of trophoblast cells in GDM mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) OGTT curves following oral gavage dose of 2 g/kg D-glucose at GD14.5.(B) AUCs of OGTT curves at GD14.5.(C, D) Fluorescence intensity and quantitative analysis of 2-NBDG in trophoblast cells by flow cytometry at GD18.5.*P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001 vs. normal group.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/87cef66d6d6363e1e4381fb7.png"},{"id":47738813,"identity":"bd900eb1-dc8d-4027-8744-5215d7c22a01","added_by":"auto","created_at":"2023-12-06 18:54:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOffspring and placental weights of GDM mice were increased\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)The offspring and placenta of normal and GDM mice. (B)Weight of offspring of mice in normal and GDM groups. (C)Placental weight of mice in normal and GDM groups. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. normal group.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/95bac3be7259eb9a728b58bc.png"},{"id":47738819,"identity":"5c045134-fb4b-4ad6-ab3a-caa3cc37d832","added_by":"auto","created_at":"2023-12-06 18:54:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":721636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDown-regulation of GLUT3 expression on trophoblast cell membranes in GDM mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Representative photomicrographs of CK7 and GLUT3 expression in immunofluorescent stained normal and GDM mouse placental tissues. (B)Representative micrographs of GLUT3 localization and expression in confocal normal and GDM mouse placental tissues. (C)qRT-PCR analysis of GLUT3.(D, E)Western blot analysis of total GLUT3 protein expression. (F, G)Wst ern blot analysis of cytoplasmic GLUT3 protein expression. (H, I)Western blot analysis of GLUT3 protein expression on cell membrane.Uncropped results of western blot were shown in supplementary file(figure S1).*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05,vs normal group.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/834ea2d3cedc2c0417759c65.png"},{"id":47738816,"identity":"980cc0a1-cc59-4796-ae0f-46f641e334b1","added_by":"auto","created_at":"2023-12-06 18:54:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":253611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep-AMPK was significantly down-regulated in the trophoblast cells of GDM mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative photomicrographs of CK7 and AMPK expression in immunofluorescently stained normal and GDM mouse placental tissues.(B)qRT-PCR analysis of AMPK.(C-E)Western blot analysis of cytoplasmic AMPK and p-AMPK protein expression.*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05,vs normal group.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/230730e3f4a09f2e7358f8eb.png"},{"id":47738815,"identity":"cb18c4f0-ceeb-4230-a592-e1538db96f2b","added_by":"auto","created_at":"2023-12-06 18:54:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction between AMPK and GLUT3 in placental tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInteraction between AMPK and GLUT3 detected by co-immunoprecipitation\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/ca3826491ccaac1451fe8dcc.png"},{"id":47739363,"identity":"5343691a-6ea8-40e4-b7cd-ac81690cc773","added_by":"auto","created_at":"2023-12-06 19:02:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":89806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAICAR reduces the blood sugar level of mice and promotes the glucose uptake capacity of trophoblast cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) OGTT curves following oral gavage dose of 2 g/kg D-glucose at GD18.5.(B) AUCs of OGTT curves at GD18.5.(C,D) Fluorescence intensity and quantitative analysis of 2-NBDG in trophoblast cells by flow cytometry at GD18.5.*P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001 GDM group vs. normal group.GDM group vs GDM+AICAR group.Normal group vs Normal+AICAR group.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/7483f9f049768d0ecc113e43.png"},{"id":47738817,"identity":"70d66e2d-0add-49b4-94a3-61ff8694581e","added_by":"auto","created_at":"2023-12-06 18:54:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":203460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAICAR up-regulated the expression levels of p-AMPK and membrane GLUT3 in mouse trophoblast cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B)qRT-PCR analysis of GLUT3 and AMPK.(C,D)Western blot analysis of total GLUT3、AMPK、P-AMPK protein expression.(E,F)Wstern blot analysis of cytoplasmic GLUT3 protein expression.(G,H)Western blot analysis of GLUT3 protein expression on cell membrane.*P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001 GDM group vs.normal group.GDM group vs GDM+AICAR group.Normal group vs Normal+AICAR group.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/58aede46234d2c7ad6710a93.png"},{"id":47739535,"identity":"68b9a15e-ade2-46b5-88dd-998cf4181fd1","added_by":"auto","created_at":"2023-12-06 19:10:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2321015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/ca520082-b98e-438e-a687-205e52f99589.pdf"},{"id":47738820,"identity":"2759938f-c729-46c3-9bd7-37946b8adfed","added_by":"auto","created_at":"2023-12-06 18:54:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":593683,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDatasetFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3680631/v1/bfa3ec902f52c54d8107e47b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Glucose uptake in trophoblasts of GDM mice is regulated by the AMPK-CLUT3 signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGestational diabetes mellitus(GDM) is one of the most common complications during pregnancy, affecting about 8\u0026ndash;25% of pregnancies \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The overall prevalence of GDM in mainland China is about 14.8%, and it has continued to increase in the past 10 years\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.GDM refers to a condition in which women without diabetes develop diabetes during pregnancy, which usually occurs in the middle or late pregnancy, and is related to insulin resistance, hyperglycemia, and maternal hyperinsulinemia\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.GDM may have an adverse effect on the health of mothers and offspring. Women with GDM are more likely to cause pregnancy complications such as miscarriage, dystocia, preeclampsia, fetal malformations, perinatal macrosomia, and neonatal hypoglycemia\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In addition, GDM may also increase the risk of type 2 diabetes, cardiovascular disease, childhood obesity, etc\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Unfortunately, the pathogenesis of GDM remains unclear, leading to a lack of targeted treatment for its underlying cause.\u003c/p\u003e \u003cp\u003eGlucose, as the main energy substrate, is essential for the growth and development of the fetus and placenta. During pregnancy, the glucose transporters(GLUTs) transport glucose from the maternal circulation to the fetus by facilitated diffusion in the placenta \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The GLUTs family includes 14 subtypes, which transport glucose and other nutrients across different tissues\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that GLUT-1, GLUT-3, GLUT-4, GLUT-8, and GLUT-12 have been detected in human placental tissues, among which GLUT1, GLUT3, and GLUT4 have been reported as the main glucose transporters\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.GLUT4 as an insulin-sensitive glucose transporter, is mainly distributed in the surrounding tissues such as fat, myocardium, and skeletal muscle, and is regulated by the insulin signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Studies have found that the expression of GLUT4 is reduced in the placenta tissue of GDM women\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The glucose metabolism of trophoblasts is possible to be regulated through the IRS1/GLUT4 signaling pathway in mice fed with a high-fat diet after FGF is given\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. At present, the uptake and transport of glucose by the placenta are mainly mediated by GLUT1 and GLUT3\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. GLUT1 is ubiquitously expressed in placental tissues and is responsible for basic glucose transport\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. The expression pattern of GLUT3 is limited, only in placental trophoblasts, and GLUT3 has a high affinity for glucose, while the affinity for other monosaccharides (such as mannose, xylose, and galactose) is much lower, indicating that GLUT3 plays a special role in the process of glucose transport in the placenta \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that in the mouse model of Intrauterine Growth Restriction(IUGR), the expression level of placental GLUT3 is down-regulated, and the glucose transported through the placenta is also reduced\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The above proofs indicate that the changes of GLUT3 in placental tissue may play an important role in glucose uptake and metabolism.\u003c/p\u003e \u003cp\u003eAdenosine 5\u0026lsquo;-monophosphate-activated protein kinase(AMPK) acts as an important energy sensor, which can be activated to promote decomposition metabolism and reduce synthetic metabolism. AMPK is a relatively complex heterotrimeric serine/threonine kinase, consisting of a catalytic (A1, A2) subunit and two regulation (B1, B2, and γ1, gamma 2 or γ3) subunits, Get active by A2 subunit 172-site threonine phosphorylation\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. AMPK controls the energy metabolism and glucose steady state of skeletal muscle, liver, adipose tissue, and pancreatic β cells. The AMPK pathway is the major target in preventing many metabolic diseases, including cancer, fatty liver diseases, and diabetes \u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Evidence suggests that activation of AMPK in skeletal muscle cells can promote GLUT4 film transition, consequently increasing glucose intake\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. However, a few studies focused on the relationship between AMPK and GLUT3 have found that AMPK in the activation of colon cancer cells can promote GLUT3 expression, and the level of GLUT3 is significantly up-regulated in the use of the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. However, it remains unclear whether the AMPK-GLUT3 regulatory axis exists in the mouse placenta. Therefore, in this study, an animal model of GDM was constructed and AICAR was used to stimulate pregnant mice to study the possible role of AMPK-GLUT3 in mouse placental tissue.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and assay kits\u003c/h2\u003e \u003cp\u003eBCA Protein Assay Kit (catalogue #: P0011), Membrane and Cytosol Protein Extraction Kit (catalogue #: P0033), Trypsin-EDTA Solution (catalogue #: C0201), BeyoColor\u0026trade; Prestained Color Protein Marker (catalogue #: P0076), SDS-PAGE Sample Loading Buffer (catalogue #: P0015L), BeyoECL Moon (catalogue #: P0018FS)were purchased from Beyotime (Shanghai, China). Phospho-AMPKa (Thr172) (40H9) Rabbit mAb(catalogue#:2535) and AMPKa Antibody(catalogue #:2532)were acquired from CST (Boston,USA). Streptozocin(catalogue #S0130), PVDF membranes (catalogue #: SAMPLG001), Protein G\u0026ndash;Agarose ༈catalogue #P7700), Dulbecco\u0026rsquo;s phosphate-buffered saline (DPBS) (catalogue #: 524650), reduced-serum\u003c/p\u003e \u003cp\u003emedium (DMEM) (catalogue #: M3817), foetal bovine serum (FBS) (catalogue #: 12103C) and Collagenase (catalogue #: C5138)were obtained from Sigma (Japan). Percoll(catalogue #: P8370), Sodium Citrate buffer(catalogue #: C1013) and Proteinase K(catalogue #: 39450-01-6) were purchased from Solarbo (beijing,China). Glut3 Antibody(catalogue #: sc-74399) was procured from SCB (Japan). D-Glucose and 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)- 186689-07-6(catalogue #: N13195)were bought from ThermoFisher (USA). AICAR༈catalogue #: S1802༉ was acquired from Selleck.cn (Shanghai, China). HRP-conjugated Affinipure Goat Anti-Mouse IgG(H\u0026thinsp;+\u0026thinsp;L)(catalogue #:SA00001-1), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L)(catalogue #:SA00001-2), Fluorescein (FITC)\u0026ndash;conjugated Affinipure Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L)(catalogue #:SA00003-2), R-PE-conjugated Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L)(catalogue #:SA00008-2), Cytokeratin 7-Specific Polyclonal Antibody(catalogue #:17513-1-AP), AMPK Alpha Monoclonal Antibody(catalogue #:66536-1-Ig) and Alpha Tubulin Polyclonal Antibody(catalogue #:11224-1-AP) were purchased from Proteintech(USA). Acryl/Bis 30% Solution(catalogue #: B546016), 1M Tris-HCl Solution, pH 6.8, Sterile(catalogue #: B548122), 4X Tris-HCl/SDS (pH 8.8)(catalogue #: B546021༉, N,N,N\u0026rsquo;,N\u0026rsquo;-Tetramethylethylenediamine (catalogue #: A610508), Ammonium persulfate(catalogue #: A100486), 10% SDS Solution(catalogue #: B548118), 10X Tris-Glycine SDS-PAGE Running Buffer(catalogue #: C520001), EZ-Buffers C 10X Western Transfer Buffer(catalogue #: C520003) and EZ-Buffers H 10X TBST Buffer(catalogue #: C520009) were acquired from Sangon Biotech (Shanghai, China). PrimeScript\u0026trade; RT Master Mix (catalogue #: RR036Q) and TB Green\u0026reg; Premix Ex Taq\u0026trade;(catalogue #: RR420Q) were obtained from Takara (Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimal and experimental design\u003c/h2\u003e \u003cp\u003eUnfertilized female C57BL/6J mice aged 6\u0026ndash;8 weeks, were purchased from Chongqing Tengxin Biological Co.Ltd, and then fed in the SPF animal laboratory of Chongqing Medical University. All procedures were conducted in accordance with ARRIVE guidelines.This study was approved by the Chongqing Medical University Ethics Committee. Mice were raised in the animal laboratory of Chongqing Medical University, on a regular 12-hour night and day cycle, and fed with a standard murine diet and water ad libitum. After being fed to around 9\u0026ndash;10 weeks, the female mice were mated with the male mice at a ratio of 1:1. Female mice were examined for the vaginal plug at 8:00 AM the following morning. The day on which a vaginal plug was detected was considered the gestation day 0.5 (GD0.5). The pregnant mice were divided into 4 groups: normal pregnancy group, GDM group, Normal\u0026thinsp;+\u0026thinsp;AICAR group, and GDM\u0026thinsp;+\u0026thinsp;AICAR group, with 3\u0026ndash;5 mice in each group. The GDM and GDM\u0026thinsp;+\u0026thinsp;AICAR groups were injected with 150mg/kg of STZ diluted in sodium citrate at GD12.5, while the Normal\u0026thinsp;+\u0026thinsp;AICAR group and GDM\u0026thinsp;+\u0026thinsp;AICAR group were injected with 250 mg/kg AICAR at GD17.5. For the oral glucose tolerance test(OGTT), each group was given 2mg/kg glucose at GD14.5 and GD18.5. Then, blood samples were collected from the tail vein at 0, 30, 60, 90, and 120 minutes to measure blood glucose levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection and preparation\u003c/h2\u003e \u003cp\u003eAll pregnant mice were euthanized at GD18.5, and offspring and placenta were collected. Phosphate-buffered saline (PBS) was pre-cooled to wash the blood of the placenta tissue until the rinse was colorless. Part of the placenta was placed in a cryotube and stored in the refrigerator at -80℃ for qRT-PCR and Western Blot experiments. The tissue sample was embedded in paraffin and stored at room temperature before immunofluorescence experiments. primary trophoblast cells were extracted from the remaining placental for 2-NBDG uptake experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2-NBDG uptake assay\u003c/h2\u003e \u003cp\u003eFirst, mouse placental tissue was added to 1 ml of collagenase IV at a concentration of 0.01 g/ml, quickly chopped, and then placed in a 37\u0026deg;C incubator for full digestion of 30 minutes. Then, 5ml of complete medium was added to stop digestion, the cell suspension was sieved with a 70um cell strainer centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded and resuspended in 5ml of stain buffer for later use. Next, percoll mixtures with concentrations of 60%, 40%, 20%, and 5% were prepared and added to a 50ml centrifuge tube in turn. The above cell suspension was slowly added to the uppermost layer. The cell suspension at 20\u0026ndash;40% density layer was collected after centrifugation at 1500 rpm for 5 minutes. Next, the mixture was resuspended in 5 ml of complete medium, and the cell pellets were collected after differential adherence for 30 minutes and resuspended and seeded in 24-well plates for 24 hours. The next day, the 24-well plate was washed twice with pre-cooled PBS, and then incubated with 100um of 2-NBDG for 30 minutes. Finally, cells were collected and the intracellular fluorescence intensity was immediately measured by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative RT-PCR\u003c/h2\u003e \u003cp\u003eTrizol was used to extract total RNA from the tissue according to the instructions, and the RNA concentration was determined. A 1 mg RNA was reversed transcribed to cDNA using the cDNA reverse transcription kit. The qRT-PCR reaction was performed with SYBRPrem ixExTaqt in the RT-PCR detection system. The gene expression levels were normalized using the housekeeping gene GAPDH. The following is the primer sequence of each gene:\u003c/p\u003e \u003cp\u003eGLUT3:Sense5\u0026prime;-GAGATGAAGGATGAGAGTGTTCGGATG-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003eAnti-sense5\u0026rsquo;-GCTGGAGGACAATGGAGATGAGAAG-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eAMPK:Sense5\u0026prime;-GCCTTGAAAGAAGTGTGTGAGAAGTTC-3\u0026rsquo;, Anti-sense5\u0026rsquo;-GTGGGTCCTGGTGGTTTCTGTTG-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eGAPDH:Sense5\u0026prime;-ATGTGTCCGTCGTGGATCTGAC-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003eAnti-sense 5\u0026prime;-AGACAACCTGGTCCTCAGTGTAG-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003eFinally, the 2-ΔΔct method was used to calculate the relative expression of sample genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and western blot\u003c/h2\u003e \u003cp\u003e30mg of placental tissue was taken from each sample, RIPA lysate was used for total protein extraction, membrane protein, and cytoplasmic protein extraction refer to kit instructions. The BCA kit was used to determine the protein concentration was determined using the BCA kit. The protein was stored in a refrigerator at -80\u0026deg;C. First, SDS-PAGE was used to separate the proteins, which were then transferred to PVDF membranes. Then, the membrane was blocked in 5% no-fat milk and hybridized overnight at 4℃ with primary antibodies, including AMPK, P-AMPK, GLUT3, and Tubulin. The next day, the membrane was washed 5 times with TBST and incubated with the corresponding secondary antibody for 1 hour. Finally, ECL was used for chemiluminescence detection, and grayscale quantitative analysis was performed by Gel-Proanlyzer software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence and confocal microscopy\u003c/h2\u003e \u003cp\u003eParaffin blocks were sliced, dewaxed, and hydrated. PBS-rinsed sections were incubated with proteinase K for 15 minutes for antigen retrieval. Sections were washed 3 times with PBS for 10 minutes each and then added 0.3% Triton-100 for 30 minutes. Sections were hybridized with primary antibody overnight at 4\u0026deg;C. The primary antibodies include AMPK, GLUT3, CK7, and Na+-K+-ATPase. Next, the sections were washed three times with PBS again, and incubated with the corresponding secondary antibodies for 1 hour. Finally, Hoechst 33442 was added and incubated with the sections for 10 minutes, and Fluorescence microscopy or confocal microscopy was performed to observe the fluorescent signal of the tissue on the slice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCo-Immunoprecipitation\u003c/h2\u003e \u003cp\u003eFor each sample, 30 mg of mouse placental tissue was placed in 1 ml of RIPA lysis buffer and lysed on ice for 30 minutes. Then, 200ul supernatant of the lysate was collected after centrifuging at 16000g at 4\u0026deg;C for 30 minutes, and stored at -80\u0026deg;C. 30ul protein A agarose beads were washed 3 times in 500ul PBS on a shaker. Next, the washed Protein A agarose beads and lysate were mixed with 2ul of AMPK or GLUT3 primary antibody, respectively, and rotated at 4\u0026deg;C overnight. The next day, the mixture was centrifuged at 3000 rpm at 4\u0026deg;C for 3 minutes, the supernatant was discarded, and the pellet was washed 3 times with 500 ul of PBS. Finally, the pellet was resuspended in 30ul of RIPA lysis buffer, and 10ul of loading buffer was added and boiled at 100\u0026deg;C for 10 minutes. The expression levels of AMPK or GLUT3 in each protein sample were detected by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAt least three independent replicates for each experiment. Measurement data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). The independent sample t-test was used to compare the two groups, and the one-way analysis of variance was used to compare the multiple groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered statistically significant. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e1. Decreased glucose uptake capacity of trophoblast cells in GDM mice\u003c/h2\u003e\n \u003cp\u003ePregnant C57 mice were injected intraperitoneally with 150 mg/kg STZ or an equal volume of PBS at GD12.5. OGTT test was performed on two groups of mice at GD14.5. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, the blood glucose of the mice in the GDM group was higher than that in the normal group at 0, 30, 60, 90, and 120 minutes after the mice were gavaged with glucose. The area under the curve of blood glucose in the GDM group was also higher than that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.A, B). In addition, the results of the 2-NBDG experiment showed that the glucose uptake capacity of the trophoblasts in the GDM group was significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.C, D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2. Offspring and placental weights of GDM mice were increased\u003c/h2\u003e\n \u003cp\u003eEuthanize mice at GD18.5 and test the offspring and placental weights of normal pregnancy and GDM mice (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.A). It was found that the weight of the offspring(P\u0026thinsp;\u0026lt;\u0026thinsp;0.001)and placenta (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001)of GDM mice were significantly higher than that of the normal pregnancy group(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.B, C).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3. Down-regulation of GLUT3 expression on trophoblast cell membranes in GDM mice\u003c/h2\u003e\n \u003cp\u003eWe found that CK7 and GLUT3 co-localized in the placental tissue of the normal and GDM groups, and the proportion of GLUT3-positive cells in the GDM group decreased by immunofluorescence (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.A). Confocal microscopy imagine showed that, GLUT3 is expressed in the cell membrane and cytoplasm of both the normal and GDM groups(Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.B). Meanwhile, the qRT-PCR experiment showed no significant difference in the transcription level of GLUT3 in the placenta tissue of the two groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. C). Previous studies have shown that GLUT3 exhibits rapid and significant membrane translocation during a marked increase in neuronal glucose uptake. Interestingly, western blot results suggested that there was no difference in the expression levels of total GLUT3 protein and cytoplasmic GLUT3 protein in the placental tissue of the normal and the GDM group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. D, E, F, G). However, the expression level of GLUT3 protein in the placental tissue of the GDM group was significantly decreased(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05,Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.H, I), which suggested that the level of GLUT3 on the trophoblast cell membranes of GDM mice was down-regulated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e4. p-AMPK was significantly down-regulated in the trophoblast cells of GDM mice\u003c/h2\u003e\n \u003cp\u003eAs a key competent molecule, activated AMPK plays an important role in glucose uptake. Immunofluorescence showed that CK7 and AMPK also co-localized in the mouse placenta(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. A), and the qRT-PCR results showed no significant difference in the transcription level of AMPK in the placenta between the two groups of mice(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. B). Phosphorylation of AMPK significantly enhances AMPK activity. Therefore, we detected the expression level of p-AMPK in placental tissue by western blot experiment. The results showed no difference in the expression level of AMPK protein in the placenta in the two groups(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.C, D), while the expression level of p-AMPK protein in the placental tissue of mice in the GDM group was significantly decreased(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.E). These results support the significant downregulation of AMPK phosphorylation in trophoblast cells of GDM mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e5. Interaction between AMPK and GLUT3 in placental tissue\u003c/h2\u003e\n \u003cp\u003eThe above evidence indicates that GLUT3, AMPK, and CK7 co-localize with each other in mouse placenta tissue. Furthermore, the plasma membrane localization of GLUT3 was significantly down-regulated in the placental tissue of GDM mice, and the phosphorylation level of AMPK was also decreased. Previous studies have also found that activation of AMPK in neurons can promote the membrane translocation of GLUT3. Hence, our study substantiated the interaction between AMPK and GLUT3 in mouse tissue(Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The findings indicated that AMPK may regulate the translocation of GLUT3 from the cytoplasm to the cell membrane through direct binding.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e6. AICAR reduces the blood sugar level of mice and promotes the glucose uptake capacity of trophoblast cells\u003c/strong\u003e\u003c/p\u003eAICAR, a well-known AMPK agonist, has been shown to reduce blood sugar in mice by activating AMPK. To investigate this further, pregnant C57 mice were administered intraperitoneal injections with 250 mg/kg AICAR or an equivalent volume of PBS at GD17.5. Subsequently, an OGTT test was performed on four groups of mice at GD18.5. The results in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA indicated that the blood glucose levels of the mice in the AICAR group were consistently lower than those of the corresponding non-AICAR group at 0, 30, 60, 90, and 120 minutes following glucose administration. Furthermore, the area under the curve for blood glucose was significantly reduced after AICAR injection (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.A, B). Additionally, the results of the 2-NBDG experiment showed that the glucose uptake capacity of the trophoblast cells also increased after AICAR injection(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.C, D).\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e7. AICAR up-regulated the expression levels of p-AMPK and membrane GLUT3 in mouse trophoblast cells\u003c/h2\u003e\n \u003cp\u003eSimilarly, the qRT-PCR findings indicated no substantial difference in the transcription levels of GLUT3 and AMPK in the placental tissue of the four groups of mice(Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e.A, B). The western Blot experiments confirmed that AICAR facilitated the phosphorylation of AMPK and the expression of GLUT3 in the mouse trophoblast cell membrane(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e.C, D, G, H).In conclusion, AICAR up-regulated the expression of GLUT3 on the trophoblast cell membrane of GDM mice by activating AMPK.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used STZ to construct a GDM mice model and found that compared with normal pregnant mice, the expression level of GLUT3 on the trophoblast membrane and the activity of AMPK in the trophoblast of GDM mice were significantly down-regulated.In vivo experiments have confirmed that AICAR can activate AMPK, thereby improving the abnormal localization of mice trophoblasts GLUT3 and increasing the glucose uptake of mice trophoblasts. In general, we speculate that AICAR increases the membrane localization of GLUT3 by upregulating the activity of AMPK in trophoblasts of GDM mice. And enhanced the glucose uptake ability of mouse trophoblast.\u003c/p\u003e \u003cp\u003eThe transfer of glucose at the interface between the mother and fetus occurs following the concentration gradient of glucose, with high concentrations in maternal blood and low concentrations in the fetus. The intrauterine hyperglycemia environment in GDM expands the glucose concentration gradient between the mother and the fetus, leading to excessive transfer of glucose from the placenta to the fetus. In turn, it affects the secretion of fetal insulin, the growth of fat cells, and the synthesis of triglycerides, ultimately leading to fetal overgrowth\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e–\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Studies have also found that GDM is related to fetal growth retardation and poor growth, An epidemiological survey in the United States shows that about 7% of the offspring of GDM females are small for gestational age(SGA)\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.In a retrospective case-control study of 1981 SGA babies conducted in China, 383 SGA babies (19.3%) were born to mothers with GDM\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. This study revealed a significant reduction in the glucose uptake capacity of placental trophoblasts in GDM mice compared to those in the normal pregnancy group. Additionally, notable increases was found in both fetal and placental weight in GDM mice. Furthermore, dysplasias were observed in the offspring of GDM mice. These findings suggest an imbalanced glucose metabolism in the placenta of GDM mice, thereby elevating the likelihood of adverse pregnancy outcomes.\u003c/p\u003e \u003cp\u003eGLUT3 in mice was first found in brain tissue and showed a high expression pattern\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Studies have found that in the hippocampus of 3XTg-AD mice, the decrease in phosphorylation of AKT in the insulin signaling pathway leads to a decrease in GLUT3 translocation. This reduction in GLUT3 on the cell membrane may lead to impaired neurons' ability to take up glucose\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Existing evidence shows that GLUT3 also plays an important role in the embryonic development of mice and the transfer of glucose from the mother to the fetus, the mouse GLUT3 mutation leads to a decrease in glucose transported through the placenta, resulting in loss of early pregnancy and fetal growth restriction in late pregnancy\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. It can be seen that GLUT3 is an important mediator of glucose transport in the placenta\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.In this study, we observed that the expression level of GLUT3 on the cell membrane in the placenta tissue of GDM mice was significantly lower than that of the normal pregnancy group, while the total expression level of GLUT3 in the two groups of mice did not change significantly. It shows that GLUT3 has abnormal localization in the placenta tissue of GDM mice. This abnormal localization may be caused by the intrauterine hyperglycemia environment.\u003c/p\u003e \u003cp\u003eAMPK regulates the activity of proteins in many important metabolic signaling pathways through the phosphorylation pathway, and is considered to be a key protein related to type 2 diabetes\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. It is worth noting that compared with the normal population, the activity of AMPK in the adipose tissue of non-pregnant obese or diabetic individuals was significantly down-regulated\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Although the exact mechanism of GDM is unclear, GDM is the precursor state of type 2 diabetes. GDM women have a 7.34 times higher risk of developing type 2 diabetes after delivery than normal pregnant women\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. More and more research focuses on the mechanism of AMPK in GDM. Studies have pointed out that the activation of AMPK can improve inflammation and insulin resistance of skeletal muscle and adipose tissue in women with GDM\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Recent studies have shown that the presence of activated AMPK in the placental tissues of humans and mice is beneficial to the differentiation of the placenta and the growth of the fetus, the expression level of the AMPK gene in the placental tissues of obese women with GDM decreases.\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e–\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Therefore, we speculate that the imbalance of glucose homeostasis in the placenta tissue of GDM mice is related to AMPK. To test this hypothesis, we constructed a GDM mice model. Although the mRNA levels of AMPK in the normal pregnancy group and GDM mice did not change significantly, Western blot experiments showed that compared with the normal pregnancy group, the phosphorylation level of AMPK in the placenta tissue of GDM mice was significantly down-regulated. Immunofluorescence showed that CK7 and AMPK also co-localized in mouse placenta. And through CO-IP detection, there is an interaction between AMPK and GLUT3 in trophoblasts. Therefore, we believe that AMPK in trophoblasts may directly interact with GLUT3 to promote the transfer of GLUT3 in the cytoplasm to the cell membrane, thereby mediating the transport of glucose, and the ingested glucose is metabolized by glycolysis for energy. The AMPK activity is inhibited in the high glucose environment caused by GDM mice, resulting in abnormal localization of GLUT3, which in turn weakens the level of glucose metabolism in the placenta itself, increases the net glucose transport from the maternal circulation to the fetal circulation, eventually affect the growth of the fetal placenta.\u003c/p\u003e \u003cp\u003eTo further verify the above findings, in this study, in vivo experiments were conducted to investigate whether AMPK activation can regulate the translocation expression of GLUT3 and glucose transport in mice placental tissues.AICAR is a commonly used AMPK agonist,it can be absorbed and metabolized by tissues in the body into ZMP, an analog of AMP, thereby activating AMPK and promoting glucose uptake by tissues\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Studies have found that AICAR stimulation in rats can increase AMPK activity, thereby improving insulin sensitivity and glucose transport in rat muscles\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e.In our study, The blood glucose levels of pregnant mice at the OGTT 5 time points and the area under the curve injected with AICAR were significantly lower than those of the non-injected group. The glucose uptake capacity of placental trophoblasts is also significantly increased after AICAR injection. It is suggested that AICAR can improve the impaired glucose uptake of placental trophoblasts and promote the glucose metabolism of the placenta itself. We also found that AICAR significantly increased the phosphorylation level of AMPK, and the expression level of GLUT3 on the cell membrane was also significantly increased. Therefore, we concluded that AICAR can activate AMPK, and promote the translocation of GLUT3 from the cytoplasm to the cell membrane in trophoblasts, thereby regulating placental glucose homeostasis.\u003c/p\u003e \u003cp\u003eIn summary, We have proved that giving AICAR to STZ-induced GDM mice can activate the trophoblasts AMPK, reversing the abnormal localization of GLUT3 in the mice trophoblasts and improving the glucose uptake capacity of the trophoblasts. In-depth discussion of the regulatory mechanism of glucose transport at the maternal-fetal interface, inhibiting excessive glucose transport to the fetus, reducing the adverse effects of the GDM intrauterine hyperglycemia environment on the fetus, and providing new intervention targets for improving the birth outcome of GDM, but this still requires greater Research to illustrate the role of AMPK-GLUT3 signal axis in regulating placental glucose homeostasis.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to conduct this study are available from the corresponding author(Juan Qiao [email protected])\u0026nbsp;upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJQ ,CTand HQ conceived the study. ZH,XL,XiaoL,RD,and PW performed the experiments and analyzed the data.ZH and XL wrote the manuscript.JQ and HQ edited the manuscript and provided funding for the work. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were conducted in accordance with ARRIVE guidelines.And this study was approved by the Chongqing Medical University Ethics Committee.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China（81901508）\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuariguata L., Linnenkamp U., Beagley J., Whiting D R., Cho N H.(2014). Global estimates of the prevalence of hyperglycaemia in pregnancy. 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J Physiol Biochem, 71(4), 703-17. doi:10.1007/s13105-015-0435-7.\u003c/li\u003e\n\u003cli\u003eCarey Erica A K., Albers Renee E., Doliboa Savannah R., Hughes Martha., Wyatt Christopher N., Natale David R C., Brown Thomas L.(2014). AMPK knockdown in placental trophoblast cells results in altered morphology and function. Stem Cells Dev, 23(23), 2921-30. doi:10.1089/scd.2014.0092.\u003c/li\u003e\n\u003cli\u003eMartino J., Sebert S., Segura M T., Garc\u0026iacute;a-Vald\u0026eacute;s L., Florido J., Padilla M C., Marcos A., Rueda R., McArdle H J., Budge H., Symonds M E., Campoy C.(2016). Maternal Body Weight and Gestational Diabetes Differentially Influence Placental and Pregnancy Outcomes. J Clin Endocrinol Metab, 101(1), 59-68. doi:10.1210/jc.2015-2590.\u003c/li\u003e\n\u003cli\u003eChoi Ran Hee., McConahay Abigail., Johnson Mackenzie B., Jeong Ha-Won., Koh Ho-Jin.(2019). Adipose tissue-specific knockout of AMPK\u0026alpha;1/\u0026alpha;2 results in normal AICAR tolerance and glucose metabolism. Biochem Biophys Res Commun, 519(3), 633-638. doi:10.1016/j.bbrc.2019.09.049.\u003c/li\u003e\n\u003cli\u003eFisher Jonathan S., Gao Jiaping., Han Dong-Ho., Holloszy John O., Nolte Lorraine A.(2002). Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab, 282(1), E18-23. doi:10.1152/ajpendo.2002.282.1.E18.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gestational diabetes mellitus, GLUT3, AMPK, Glucose uptake, AICAR","lastPublishedDoi":"10.21203/rs.3.rs-3680631/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3680631/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e: GDM as a metabolic disease during pregnancy, regulates GLUT3 translocation by AMPK, thereby affecting glucose uptake in trophoblasts. It provides a new research idea and therapeutic target for alleviating intrauterine hyperglycemia in GDM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: STZ was used to construct GDM mice, inject AICAR into pregnant mice, and observe fetal and placental weight; flow cytometry was employed for the detection of glucose uptake by primary trophoblast cells; immunofluorescence was applied to detect the localization of GLUT3 and AMPK in placental tissue; Cocofal microscope was used to detect the localization of GLUT3 in trophoblast cells;qRT-PCR and Western blot experiments were carried out to detect the expression levels of GLUT3 and AMPK in placental tissue; CO-IP was utilized to detect the interaction of GLUT3 and AMPK.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Compared with the normal pregnancy group, the weight of the fetus and placenta of GDM mice increased (P\u0026lt;0.001), and the ability of trophoblasts to take up glucose decreased (P\u0026lt;0.001).In addition, AMPK activity in trophoblasts and membrane localization of GLUT3 in GDM mice were down-regulated compared with normal pregnant mice (P\u0026lt;0.05). There is an interaction between GLUT3 and AMPK. Activating AMPK in trophoblasts can up-regulate the expression of GLUT3 membrane protein in trophoblasts of mice (P\u0026lt;0.05) and increase the glucose uptake of trophoblasts (P\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e: Inhibition of AMPK activity in GDM mice results in aberrant localization of GLUT3, which in turn attenuates glucose uptake by placental trophoblast cells.AICAR activates AMPK to increase the membrane localization of GLUT3 and improve the glucose uptake capacity of trophoblasts.\u003c/p\u003e","manuscriptTitle":"Glucose uptake in trophoblasts of GDM mice is regulated by the AMPK-CLUT3 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-06 18:54:08","doi":"10.21203/rs.3.rs-3680631/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-04T16:57:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-06T00:24:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"482bef1c-b7e9-4dbe-9d5e-38c4c8d081d8","date":"2024-01-31T18:26:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-26T14:55:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a1dd9a3d-d9ad-4bcc-9609-17b194800ad9","date":"2024-01-15T12:12:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-06T20:05:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-02T09:32:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-12-05T05:44:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-05T05:41:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-11-29T08:50:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9874165-39fa-418b-be7f-a26148d85ec1","owner":[],"postedDate":"December 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-08T15:31:28+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-06 18:54:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3680631","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3680631","identity":"rs-3680631","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}