RÉSUMÉ
INTRODUCTION: Gestational diabetes mellitus (GDM) is the most common metabolic disease in pregnancy. However, studies of activating molecule of Beclin1-regulated autophagy (Ambra1) affecting the insulin substrate receptor 1/phosphatidylinositol 3 kinase/protein kinase B (IRS-1/PI3K/Akt) signalling pathway in GDM have not been reported. The aim of the study was to detect the difference of Ambra1 expression in the placenta of normal pregnant women and GDM patients. MATERIAL AND METHODS: An in vitro model of gestational diabetes mellitus was established by inducing HTR8/Svneo cells from human chorionic trophoblast layer with high glucose. The changes of cell morphology were observed by inverted microscope, and the expression levels of Ambra1 gene and protein in model cells were detected. After this, Ambra1 gene was silenced by shRNA transfection, and PI3K inhibitor was added to detect changes in Ambra1, autophagy, and insulin (INS) signalling pathways. RESULTS: The protein expression levels of Ambra1, Bcl-2 interacting protein (Beclin-1), and microtubule-associated proteins 1A/1B light chain 3B (LC3-II) in the placentas of GDM pregnant women were higher than those of normal pregnant women. High glucose induces morphological changes in HTR8/Svneo cells and increases Ambra1 transcription and translation levels. sh-Ambra1 increased survival of HTR8/SvNEO-HG cells and inhibited Ambra1, Beclin1, and LC3-II transcription and translation levels. Also, sh-Ambra1 increased IRS-1/PI3K/Akt protein phosphorylation levels and inhibited the IRS-1/PI3K/Akt signalling pathway and its resulting autophagy. CONCLUSIONS: sh-Ambra1 increased IRS-1/PI3K/Akt protein phosphorylation levels to reduce autophagy in gestational diabetes.
Sujet(s)
Diabète gestationnel , Femelle , Humains , Grossesse , Autophagie , Bécline-1 , Diabète gestationnel/métabolisme , Glucose/métabolisme , Insuline/métabolisme , Phosphatidylinositol 3-kinases/métabolisme , Protéines proto-oncogènes c-akt/métabolismeRÉSUMÉ
The relationship between tyrosine phosphorylation (TP) and protein expression of insulin receptor (InsR) and insulin resistance (IR) in patients with gestational diabetes mellitus (GDM) was investigated. The InsR expression and TP in skeleton muscle tissue were determined by Western blotting and immunoprecipitation in women with GDM (GDM group, n=22), normal pregnant women (normal pregnancy group, n=22) and normal non-pregnant women (normal non-pregnant group, n=13). Fasting plasma glucose (FPG) and fasting insulin (FINS) were measured by oxidase assay and immunoradioassay. The results showed that the levels of FPG (5.61±0.78 mmol/L), FINS (15.42±5.13 mU/L) and Homeostasis model assessment-IR (HOMA-IR) (1.21±0.52) in GDM group were significantly higher than those in normal pregnancy group (4.43±0.46 mmol/L, 10.56±3.07 mU/L and 0.80±0.31 respectively) (P<0.01). The levels of FINS and HOMA-IR in normal pregnancy group were significantly higher than those in normal non-pregnant group (7.56±2.31 mU/L and 0.47±0.26 respectively) (P<0.01). There was no significant difference in the InsR expression level among the three groups (P>0.05). TP of InsR with insulin stimulation was significantly decreased in GDM group (0.20±0.05) as compared with normal pregnancy group (0.26±0.06) (P<0.01). TP of InsR with insulin stimulation in normal pregnancy group was lower than that in normal non-pregnant group (0.31±0.06) (P<0.01). TP of InsR with insulin stimulation was negatively related with HOMA-IR in GDM group (r=-0.525, P<0.01). There was no correlation between the protein expression of InsR and HOMA-IR in GDM group (r=-0.236, P>0.05). It was suggested that there is no significant correlation between the protein expression of InsR in skeletal muscle and IR in GDM, but changes in TP of InsR are associated with IR in GDM.
Sujet(s)
Diabète gestationnel/métabolisme , Insulinorésistance , Récepteur à l'insuline/métabolisme , Tyrosine/métabolisme , Adulte , Glycémie/métabolisme , Technique de Western , Diabète gestationnel/sang , Jeûne/sang , Femelle , Humains , Insuline/sang , Muscles squelettiques/métabolisme , Phosphorylation , Grossesse , Dosage radioimmunologiqueRÉSUMÉ
OBJECTIVE: To investigate the expression of phosphatidylinositol 3-kinase (PI-3K) in adipose tissue of polycystic ovary syndrome patients (PCOS), and explore molecular mechanisms of insulin resistance (IR) in PCOS. METHODS: Samples from patients with PCOS with IR (n = 19), PCOS without IR (n = 10) and controls (n = 15) were collected. Serum fasting insulin (FIN) and fasting plasma glucose (FPG) were measured. Insulin resistance index was calculated using homeostasis model assessment (HOMA) to analyze the relationship between these markers and IR. Western blot technique was used to detect the PI-3K p85 subunit. Gene expression of PI-3K p85 subunit was detected by reverse transcription polymerase chain reaction (RT-PCR) method. Kinase activity was detected by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. RESULTS: (1) The levels of FIN [(25.2 +/- 3.8) mU/L] and HOMA-IR (1.6 +/- 0.3) in PCOS with IR were significantly higher than those in PCOS without IR [(13.4 +/- 3.8) mU/L, 0.9 +/- 0.3] and controls [(9.5 +/- 2.6) mU/L, 0.5 +/- 0.3; all P < 0.05). (2) There was no significant difference in the protein (0.65 +/- 0.10) and gene expression (0.92 +/- 0.12) of PI-3K p85 subunit in PCOS with IR compared with PCOS without IR (0.72 +/- 0.10, 1.01 +/- 0.10) and control groups (0.73 +/- 0.14, 1.00 +/- 0.12; P > 0.05). (3) PI-3K activity in PCOS with IR (81%) and PCOS without IR (89%) was significantly decreased (P < 0.01, P < 0.05) and negatively correlated with HOMA-IR (r = -0.69, P < 0.01; r = -0.62, P < 0.05). CONCLUSIONS: No significant difference in the protein and gene expression of PI-3K p85 subunit in PCOS with IR is found. The decreased PI-3K activity may lead to IR of PCOS.
Sujet(s)
Tissu adipeux/enzymologie , Insulinorésistance , Phosphatidylinositol 3-kinases/métabolisme , Syndrome des ovaires polykystiques/enzymologie , Adulte , Marqueurs biologiques/sang , Glycémie/analyse , Technique de Western , Jeûne/sang , Femelle , Homéostasie , Humains , Insuline/sang , Phosphatidylinositol 3-kinases/génétique , Syndrome des ovaires polykystiques/sang , Syndrome des ovaires polykystiques/physiopathologie , ARN messager/génétique , ARN messager/métabolisme , RT-PCRRÉSUMÉ
OBJECTIVE: To explore molecular mechanisms of insulin resistance of polycystic ovary syndrome (PCOS) by determining the tyrosine phosphorylation and protein expression of insulin receptor substrate-2 (IRS-2) in adipose tissue from patients with PCOS. METHODS: Serum and subcutaneous adipose tissue samples from patients with PCOS with insulin resistance (n = 19), PCOS without insulin resistance (n = 17) and controls (n = 20) were collected. The expression of IRS-2 in adipose tissue was assessed by Western blot. Immunohistochemistry was used to detect the distribution of IRS-2 in adipose tissues of all patients. The tyrosine phosphorylation of IRS-2 was measured by immunoprecipitation and enhanced chemiluminescent immunoblotting technique. RESULTS: (1) There was no significant difference of the protein expression of IRS-2 in PCOS with insulin resistance 1.15 +/- 0.26 compared to those in PCOS without insulin resistance 1.13 +/- 0.26 and control group 1.00 +/- 0.25 (P > 0.05); (2) The tyrosine phosphorylation of IRS-2 was significantly decreased in PCOS with insulin resistance 0.77 +/- 0.16 compared to that of PCOS without insulin resistance 0.91 +/- 0.25 and control groups 1.00 +/- 0.12 (P < 0.05). There was no significant difference between PCOS without insulin resistance and control groups (P > 0.05). CONCLUSIONS: The decrease of tyrosine phosphorylation of IRS-2 in PCOS patients, which induces impairment of the insulin signal pathway, may be one of the mechanisms leading to insulin resistance.
Sujet(s)
Tissu adipeux/métabolisme , Insulinorésistance , Protéines et peptides de signalisation intracellulaire/métabolisme , Phosphoprotéines/métabolisme , Syndrome des ovaires polykystiques/métabolisme , Tyrosine/métabolisme , Adulte , Glycémie/analyse , Technique de Western , Femelle , Hormone folliculostimulante/sang , Humains , Immunohistochimie , Insuline/sang , Substrats du récepteur à l'insuline , Hormone lutéinisante/sang , Phosphorylation , Syndrome des ovaires polykystiques/physiopathologie , Transduction du signalRÉSUMÉ
OBJECTIVE: To investigate the tyrosine phosphorylation and protein expression of insulin receptor substrate-1 in adipose tissue from patients with polycystic ovary syndrome, and explore molecular mechanisms of insulin resistance of PCOS. METHODS: Samples from patients with PCOS with insulin resistance (group A, n = 19), PCOS without insulin resistance (group B, n = 10) and controls group (n = 15) were collected. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone (T) were measured by chemiluminescence assay. Fasting insulin (FIN) was measured by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Insulin resistance index was calculated using homeostasis model assessment (HOMA) to analyze the relationship between these markers and insulin resistance. The amount of insulin receptor substrate-1 in adipose tissue was assessed by western blot. The tyrosine phosphorylation of IRS-1 was measured by immunoprecipitation. RESULTS: (1) The levels of serum LH (15.8 +/- 2.8) U/L, LH/FSH 2.8 +/- 0.6, T (4.3 +/- 0.9) nmol/L, FIN (25.2 +/- 3.8) mU/L and HOMA IR (1.56 +/- 0.25) in group A were significantly higher than those of group B (13.9 +/- 1.9) U/L, 2.3 +/- 0.4, (3.6 +/- 0.4) nmol/L, (13.4 +/- 3.8) mU/L, 0.87 +/- 0.28 and control group (7.3 +/- 2.1) U/L, 1.3 +/- 0.3, (0.9 +/- 0.2) nmol/L, (9.5 +/- 2.6) mU/L, 0.50 +/- 0.30 (all P < 0.05); (2) The protein expression and tyrosine phosphorylation of IRS-1 in group A (690 +/- 19 and 528 +/- 72 respectively) were significantly lower than those in group B (892 +/- 31, 801 +/- 64) and control group (988 +/- 29, 1139 +/- 124) (P < 0.05, and P < 0.01 respectively). (3) Insulin resistance index in group A and group B were negatively related with protein expression and tyrosine phosphorylation (r = -0.52, P < 0.05; r = -0.61, P < 0.01 and r = -0.60, P < 0.05; r = -0.63, P < 0.05). CONCLUSIONS: The signal transduction malfunction because of protein expression and tyrosine phosphorylation changes of IRS-1 in adipose tissue from polycystic ovary syndrome patients may be one of the mechanisms leading to insulin resistance.