Silencing TRIM29 Sensitizes Non-small Cell Lung Cancer Cells to Anlotinib by Promoting Apoptosis via Binding RAD50.

BACKGROUND: Previous studies have proposed that the transcriptional regulatory factor tripartite motif containing 29 (TRIM29) is involved in carcinogenesis via binding with nucleic acid. TRIM29 is confirmed to be highly expressed when the cancer cells acquire therapy-resistant properties. We noticed that TRIM29 levels were significantly increased in anlotinib-resistant NCI-H1975 (NCI-H1975/AR) cells via mining data information from gene expression omnibus (GEO) gene microarray (GSE142031; log2 fold change > 1, p < 0.05). OBJECTIVE: Our study aimed to investigate the function of TRIM29 on the resistance to anlotinib in non-small cell lung cancer (NSCLC) cells, including NCI-H1975 and A549 cells. METHODS: Real-time RT-PCR and western blot were used to detect TRIM29 expression in anlotinib-resistant NSCLC (NSCLC/AR) cells. Apoptosis were determined through flow cytometry, acridine orange/ethidium bromide staining as well as western blot. ELISA was used to measure the content of C-X3-C motif chemokine ligand 1. Co-Immunoprecipitation assay was performed to verify the interaction between TRIM29 and RAD50 double-strand break repair protein (RAD50). RESULTS: TRIM29 expression was shown to be elevated in the cytoplasm and nucleus of NSCLC/AR cells compared to normal NSCLC cells. Next, we demonstrated that TRIM29 knockdown facilitated apoptosis and enhanced the sensitivity to anlotinib in NSCLC/AR cells. Based on the refined results citing from the database BioGRID, it was proved that TRIM29 interacted with RAD50. Herein, RAD50 overexpression diminished the pro-apoptotic effect induced by silencing TRIM29 in anlotinib-resistant A549 (A549/AR) cells. CONCLUSION: Finally, we concluded that the increased sensitivity to anlotinib in NSCLC/AR cells was achieved by knocking down TRIM29, besides, the positive effects of TRIM29 knockdown were attributed to the promotion of apoptosis via binding to RAD50 in NSCLC/AR cell nucleus. Therefore, TRIM29 might become a potential target for overcoming anlotinib resistance in NSCLC treatment.

Circ-CCNB1 Modulates Trophoblast Proliferation and Invasion in Spontaneous Abortion by Regulating miR-223/SIAH1 axis.

CONTEXT: Spontaneous abortion (SA) is a common disorder in early pregnancy. Circular RNAs (circRNAs) have been reported to exert important regulatory effects on trophoblast function and embryo development. OBJECTIVE: The aim of this study was to explore whether and how circRNAs regulate trophoblast function in SA during early pregnancy. METHODS: Cell proliferation, 5-bromo-2-deoxyuridine (BrdU) staining, Transwell, immunofluorescence, Western blot, RNA pull-down, and dual luciferase reporter assays were performed to investigate the effect of circRNA cyclin B1 (circ-CCNB1) on trophoblast function in HTR-8/SVneo and JEG-3 cells. RESULTS: An in vitro study demonstrated that upregulation of circ-CCNB1 significantly inhibited trophoblast proliferation and invasion compared with the controls using HTR-8/SVneo and JEG-3 cells, respectively. Moreover, miR-223 was downregulated in the villous tissues of patients with SA and was further predicted and shown to negatively interact with circ-CCNB1, which is involved in trophoblast proliferation and invasion. Using bioinformatics tools and subsequent RNA pull-down and dual luciferase assays, we found that miR-223 directly targets seven in absentia homolog-1 (SIAH1) and that upregulation of miR-223 decreased circ-CCNB1-induced SIAH1 expression levels in HTR-8/SVneo cells. Interestingly, upregulation of circ-CCNB1 suppressed trophoblast proliferation and invasion through inhibition of CCNB1 nuclear translocation induced by SIAH1. Downregulation of SIAH1 enhanced circ-CCNB1-suppressed CCNB1 nuclear protein expression in trophoblast cells. CONCLUSION: Circ-CCNB1 served as a modulator of trophoblast proliferation and invasion by sponging miR-223, thus forming a regulatory network of circ-CCNB1/miR-223/SIAH1 in modulating CCNB1 nuclear translocation, which enabled us to elucidate the molecular mechanisms involved in normal embryo implantation or in SA.

All-trans retinoic acid impairs glucose-stimulated insulin secretion by activating the RXR/SREBP-1c/UCP2 pathway.

Diabetes is often associated with vitamin A disorders. All-trans retinoic acid (ATRA) is the main active constituent of vitamin A. We aimed to investigate whether ATRA influences diabetic progression and its mechanisms using both Goto-Kazizazi (GK) rats and INS-1 cells. Rat experiments demonstrated that ATRA treatment worsened diabetes symptoms, as evidenced by an increase in fasting blood glucose (FBG) levels and impairment of glucose homeostasis. Importantly, ATRA impaired glucose-stimulated insulin secretion (GSIS) and increased the expression of sterol regulatory element-binding protein 1c (SREBP-1c) and uncoupling protein 2 (UCP2) in the rat pancreas. Data from INS-1 cells also showed that ATRA upregulated SREBP-1c and UCP2 expression and impaired GSIS at 23 mM glucose. Srebp-1c or Ucp2 silencing attenuated GSIS impairment by reversing the ATRA-induced increase in UCP2 expression and decrease in ATP content. ATRA and the retinoid X receptor (RXR) agonists 9-cis RA and LG100268 induced the gene expression of Srebp-1c, which was almost completely abolished by the RXR antagonist HX531. RXRα-LBD luciferase reporter plasmid experiments also demonstrated that ATRA concentration-dependently activated RXRα, the EC50 of which was 1.37 µM, which was lower than the ATRA concentration in the pancreas of GK rats treated with a high dose of ATRA (approximately 3 µM), inferring that ATRA can upregulate Srebp-1c expression in the pancreas by activating RXR. In conclusion, ATRA impaired GSIS partly by activating the RXR/SREBP-1c/UCP2 pathway, thus worsening diabetic symptoms. The results highlight the roles of ATRA in diabetic progression and establish new strategies for diabetes treatment.

Bile duct ligation causes opposite impacts on the expression and function of BCRP and P-gp in rat brain partly via affecting membrane expression of ezrin/radixin/moesin proteins.

Breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are co-located at blood-brain barrier (BBB) cells, preventing their substrates from entering brain. Accumulating evidence demonstrates that liver failure impairs P-gp and BCRP expression and function in the brain. In the current study, we investigated how liver failure influenced the expression and function of brain BCRP and P-gp in rats subjected to bile duct ligation (BDL). The function of BCRP, P-gp and BBB integrity was assessed using distribution of prazosin, rhodamine 123 and fluorescein, respectively. We showed that BDL significantly decreased BCRP function, but increased P-gp function without affecting BBB integrity. Furthermore, we found that BDL significantly downregulated the expression of membrane BCRP and upregulated the expression of membrane P-gp protein in the cortex and hippocampus. In human cerebral microvascular endothelial cells, NH4Cl plus unconjugated bilirubin significantly decreased BCRP function and expression of membrane BCRP protein, but upregulated P-gp function and expression of membrane P-gp protein. The decreased expression of membrane BCRP protein was linked to the decreased expression of membrane radixin protein, while the increased expression of membrane P-gp protein was related to the increased location of membrane ezrin protein. Silencing ezrin impaired membrane location of P-gp, whereas silencing radixin impaired membrane location of BCRP protein. BDL rats showed the increased expression of membrane ezrin protein and decreased expression of membrane radixin protein in the brain. We conclude that BDL causes opposite effects on the expression and function of brain BCRP and P-gp, attributing to the altered expression of membrane radixin and ezrin protein, respectively, due to hyperbilirubinemia and hyperammonemia.

Short-chain fatty acids exert opposite effects on the expression and function of p-glycoprotein and breast cancer resistance protein in rat intestine.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are involved in intestinal barrier. Short-chain fatty acids (SCFAs) play important roles in maintaining intestinal barrier. In this study we explored how SCFAs affected the expression and function of intestinal P-gp and BCRP in rats. Rats received 150 mM acetate, propionate or butyrate in drinking water for 4 weeks. In SCFA-treated rats, the expression and function of intestinal P-gp were decreased, but those of intestinal BCRP were increased; intestinal p-p65 was also decreased, which was positively related to P-gp protein expression. Among the three SCFAs tested, butyrate exhibited the strongest induction or inhibitory effect, followed by propionate and acetate. Similar results were observed in mouse primary enterocytes and Caco-2 cells treated with acetate (5 mM), propionate (2 mM), or butyrate (1 mM). In Caco-2 cells, addition of butyrate, vorinostat, and valproate (two classic HDAC inhibitors), Bay117082 (selective inhibitor of NF-κB activation) or NF-κB p65 silencing significantly decreased the expression of P-gp and the level of phosphorylated p65 (p-p65). Furthermore, butyrate attenuated the expression of P-gp and p-p65 induced by TNF-α (NF-κB activator) and theophylline (HDAC activator). However, vorinostat, valproate, Bay117082, TNF-α or p65 silencing hardly affected BCRP protein expression. But GW9662 (selective PPARγ antagonist) or PPARγ silencing abolished BCRP induction by butyrate and troglitazone (PPARγ agonist). SCFAs-treated rats showed higher intestinal protein expression of PPARγ, which was positively related to BCRP protein expression. Butyrate increased plasma exposure of fexofenadine but decreased that of rosuvastatin following oral dose to rats. In conclusion, SCFAs exert opposite effects on the expression and function of intestinal P-gp and BCRP; butyrate downregulated P-gp expression and function possibly via inhibiting HDAC/NF-κB pathways; butyrate induced BCRP expression and function partly via PPARγ activation.

Glycemic control and comprehensive metabolic risk factors control in older adults with type 2 diabetes.

BACKGROUND: Older adults with type 2 diabetes are prone to multiple metabolic abnormalities. However, data from these patients on comprehensive metabolic risk factors control are limited. METHODS: The present study included 2736 older adults aged 60 to 90 years with type 2 diabetes from 114 hospitals across 22 provinces in China. Metabolic abnormalities, including hypertension, dyslipidemia, hyperuricemia, and obesity, were recorded. Comprehensive metabolic risk factors control included the control of hemoglobin A1c (HbA1c) level, blood pressure, serum lipids level, serum uric acid level, and body mass index. The target glycemic control was defined as HbA1c <7%. RESULTS: The proportion of older adults who achieved the HbA1c target was 23.0%. The glycemic control rate increased with the number of metabolic abnormalities increased. The patients who had all four metabolic abnormalities had 4.05 times (95% confidence interval: 2.16, 7.61) the odd to meet glycemic target than those with none of metabolic abnormalities. However, only 4.6% of patients met the targets for all 5 metabolic risk factors. The comprehensive rate of all 5 factors in control decreased from 13.4% to 0% with the number of metabolic abnormalities increased. CONCLUSION: The glycemic control rate and the comprehensive metabolic risk factors control rate were 23.0% and 4.6%, respectively. As the number of metabolic abnormalities increased, the number of risk factor targets achieved decreased. Our findings suggest that a strategy for comprehensive control is urgently needed in older adults with type 2 diabetes, especially in those with co-existing metabolic abnormalities.

Estradiol promotes trophoblast viability and invasion by activating SGK1.

BACKGROUND: 17ß-Estradiol (E2) is a critical regulator of trophoblast function during pregnancy. Serum- and glucocorticoid-inducible kinase (SGK1) has been shown to regulate specific cellular targets downstream of E2. However, whether and how SGK1 directly mediates the regulatory effects of E2 on trophoblasts functions remain unknown. METHODS: SGK1 expression in human villous samples and serum E2 levels were measured in women with early pregnancy loss (EPL) and healthy pregnant women. The effect of E2 on SGK1 regulation was assessed using luciferase reporter gene assay and Chromatin Immunoprecipitation assay. The mediation of regulatory effects of E2 by SGK1 on trophoblast functions including cell viability, invasion and related signaling molecules such as B cell leukemia/lymphoma 6, E-cadherin, matrix metalloproteinase 2, α-ENaC, vascular endothelial growth factor, and the phosphorylation status of FOXO1 and AKT were evaluated in HTR8/SVneo cells transfected with SGK1 knockdown plasmid with/without E2 treatment. RESULTS: SGK1 protein levels in human villous samples and serum E2 levels were decreased in patients with EPL compared to controls. E2 (10 nM) increased SGK1 promoter activity directly through estrogen receptor. E2-activated SGK1 enhanced cell viability, invasion and downstream targets in trophoblast cells. SGK1 knockdown abrogated the above responses to E2 treatment. CONCLUSIONS: SGK1 mediates the effects of E2 on trophoblast viability and invasion, suggesting that SGK1 acts as a key node in regulating the cross-talk at the feto-maternal interface during the development of placenta and might be a potential therapeutic target for EPL.

Shikonin sensitizes A549 cells to TRAIL-induced apoptosis through the JNK, STAT3 and AKT pathways.

BACKGROUND: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, can selectively kill cancer cells with little or no cytotoxicity toward normal human cells and is regarded as a potential relatively safe antitumor drug. However, some cancer cells are resistant to TRAIL-induced apoptosis. Thus, reagents that potentiate TRAIL-induced cytotoxicity are needed. Herein, we investigated whether shikonin, a natural compound from the root of Lithospermum erythrorhizon, can sensitize TRAIL-resistant cells to TRAIL-induced cytotoxicity. RESULTS: The viability of A549 cells, which were resistant to TRAIL, was significantly decreased after treatment with TRAIL followed by shikonin. The underlying mechanisms by which shikonin sensitizes cells to TRAIL-induced cytotoxicity were also examined. Combined treatment with shikonin and TRAIL activated the caspase and JNK pathways, inhibited the STAT3 and AKT pathways, downregulated the expression of Mcl-1, Bcl-2, Bcl-xL, c-FLIP and XIAP and upregulated the expression of Bid. CONCLUSIONS: In conclusion, the results indicated that shikonin sensitized resistant cancer cells to TRAIL-induced cytotoxicity via the modulation of the JNK, STAT3 and AKT pathways, the downregulation of antiapoptotic proteins and the upregulation of proapoptotic proteins.

A Critical Role of Autophagy in Regulating Microglia Polarization in Neurodegeneration.

Neuroinflammation and autophagy dysfunction are closely related to the development of neurodegeneration such as Parkinson's disease (PD). However, the role of autophagy in microglia polarization and neuroinflammation is poorly understood. TNF-α, which is highly toxic to dopaminergic neurons, is implicated as a major mediator of neuroinflammation in PD. In this study, we found that TNF-α resulted in an impairment of autophagic flux in microglia. Concomitantly, an increase of M1 marker (iNOS/NO, IL-1ß, and IL-6) expression and reduction of M2 marker (Arginase1, Ym1/2, and IL-10) were observed in TNF-α challenged microglia. Upregulation of autophagy via serum deprivation or pharmacologic activators (rapamycin and resveratrol) promoted microglia polarization toward M2 phenotype, as evidenced by suppressed M1 and elevated M2 gene expression, while inhibition of autophagy with 3-MA or Atg5 siRNA consistently aggravated the M1 polarization induced by TNF-α. Moreover, Atg5 knockdown alone was sufficient to trigger microglia activation toward M1 status. More important, TNF-α stimulated microglia conditioned medium caused neurotoxicity when added to neuronal cells. The neurotoxicity was further aggravated when Atg5 knockdown in BV2 cells but alleviated when microglia pretreatment with rapamycin. Activation of AKT/mTOR signaling may contribute to the changes of autophagy and inflammation as the AKT specific inhibitor perifosine prevented the increase of LC3II (an autophagic marker) in TNF-α stimulated microglia. Taking together, our results demonstrate that TNF-α inhibits autophagy in microglia through AKT/mTOR signaling pathway, and autophagy enhancement can promote microglia polarization toward M2 phenotype and inflammation resolution.

Controlled Construction of Cu(I) Sites within Confined Spaces via Host-Guest Redox: Highly Efficient Adsorbents for Selective CO Adsorption.

Here, we designed a double-solvent/host-guest redox combined strategy to construct Cu+ sites in metal-organic frameworks (MOFs) for the first time. As a proof of concept, a representative MOF MIL-100(Fe) with tunable valence states of cations was employed as the host. The combined strategy realizes selective introduction of Cu2+ precursors to the interior pores of MIL-100(Fe), remarkably minimizing the aggregation of Cu2+ and subsequently formed Cu+ species. Owing to the proper reducibility of in situ formed Fe2+ in the frameworks, controlled conversation of Cu2+ to Cu+ with ∼100% yield is achieved in the absence of any additional reducing agents. These characteristics make the obtained materials Cu+-modified MIL-100(Fe) highly active in selective CO adsorption. The CO adsorption capacity is up to 3.75 mmol·g-1 at 298 K and 1 bar, which is superior to all other Cu+-containing adsorbents reported so far such as CuCl/activated carbon (2.5 mmol·g-1), CuCl/γ-Al2O3 (1.0 mmol·g-1), and CuCl/SBA-15 (0.50 mmol·g-1). The same adsorbent also exhibits quite high selectivity of CO over N2, and the ideal adsorption solution theory selectivity reaches 424. The outstanding CO adsorption performance make the present adsorbents great potential in separation of CO from various mixtures.

Cloning of neuromedin B and its receptor in the rabbit and generating a polyclonal antibody to the neuromedin B protein.

Neuromedin B (NMB) is a highly conserved bombesin-related neuropeptide found in mammals. Neuromedin B (NMB) executes its effect by binding to the cell surface receptor, neuromedin B receptor (NMBR). In this study, we cloned the rabbit NMB and NMBR genes. The similarity and phylogenetic analyses of NMB and NMBR gene sequences were performed. The expression of NMB and NMBR mRNA in the rabbit was investigated using real-time RT-PCR. Our bioinformatic analysis demonstrated that the cloned rabbit NMB precursor cDNA encoded Gly-His-Phe-Met-NH2 amino acids at the C-terminus, and that its receptor possessed typical transmembrane features. The NMB mRNA was highly expressed in the CNS, while the NMBR mRNA was widely expressed in many tissues, with the highest expression in the gastrointestinal tract. The studies on the NMB distribution and function are limited by the lack of a specific antibody to this neuropeptide. In this paper, polyclonal NMB antibody was generated in mice. Western blotting analysis revealed that the prepared antibody could specifically recognize the recombinant and the endogenous NMB proteins. Immunohistochemistry analysis indicated that the NMB protein was localized in the cytoplasm of the pituitary cells. The existence of NMB protein in the hypothalamic-pituitary-gonadal axis suggests that NMB might function in rabbit reproduction.

Notch signaling molecules as prognostic biomarkers for non-small cell lung cancer.

Notch family proteins have been reported to be associated with the initiation and development of various types of tumors. The present study used a prospective design to investigate the role of Notch proteins as novel biomarkers that are capable of predicting the survival outcome for patients with non-small cell lung cancer (NSCLC). The protein expression of Notch 1, Notch 3 and their ligands, Jagged 1 and Delta-like 4, was examined using immunohistochemistry in NSCLC tissues and adjacent non-cancerous lung tissues from 101 patients who underwent surgical treatment. The expression was also correlated with clinicopathological parameters and overall survival (OS). High Notch 1 protein expression was observed in 55.4% (56/101) of NSCLC samples and high Notch 3 expression was observed in 53.5% (54/101). The nuclear expression of Notch 3 was significantly associated with the lymph node status (P=0.0026) and tumor-node-metastasis (TNM) stage (P<0.0001), while the coexpression of Notch 1 plus Notch 3 was associated with lymph node status (P=0.0056), TNM stage (P=0.0001) and the histological grading (P=0.0359). In the survival analyses, the high expression of Notch 1 and Notch 3 exhibited an additive effect toward a poorer OS compared with a subtype with low coexpression for the two proteins (P<0.001), with high nuclear Notch 3 expression in the NSCLC patients maintaining independent prognostic significance for the outcome on multivariate analysis. These data further demonstrate a central role for Notch signaling in NSCLC and the significance of Notch 3 as a prognostic indicator of a poorer survival for patients with resected NSCLC.

[Analysis on the relationship between p57 and cholesterol in cellular membrane].

AIM: To investigate the relationship between p57 and cholesterol in cellular membrane under physiological conditions. METHODS: Different concentrations of mbetaCD were used to extract cholesterol from cellular membrane of U937 cells. The cellular membrane was prepared by ultracentrifugation and the content change of p57 in cellular membrane component was measured by Western blot analysis. Discontinuous sucrose density gradient centrifugation was used to prepare lipid rafts and the content of p57 in them was measured by Western blot analysis. RESULTS: The degradation of cholesterol level in cellular membrane didn't lead to obvious content change of p57 in cellular membrane component. Furthermore, no p57 protein was found in lipid rafts. CONCLUSION: No relationship between p57 and cholesterol in cellular membrane was found in the physiological condition, which is obviously different from that in pathological condition caused by the invasion of pathogenic mycobacteria.

[Association between metabolic syndrome and the 10 years mortality of cerebro-cardiovascular diseases in the senile population].

OBJECTIVE: To assess the prevalence of metabolic syndrome (MS) and its association with mortality of cerebro-cardiovascular diseases in senile population. METHODS: Data were collected from 1926 people aged 60 and over, who took part in routine health examination in our hospital from 1996 to 1997. All subjects were followed up for 10 years. MS was diagnosed by using the definition recommended by Chinese Diabetic Society in 2004. Cox-proportional hazards models were used in survival analyses and to calculate the relative risk (RR) of cerebro-cardiovascular diseases mortality. RESULTS: The prevalence of MS was 25.03% (n = 482, Group 2) in this population. The 10 year mortality of cerebro-cardiovascular diseases was significantly higher (6.82/1000-person year vs. 2.55/1000-person year, P < 0.05) and the cumulative survival rate was significantly lower (92.46%vs. 97.14%, P < 0.05) in group 2 compared that in group 1 (non-MS, n = 1444). Compared with group 1, RR of cerebro-cardiovascular diseases mortality was 2.52 (95% CI 1.367 - 4.661, P < 0.05) in group 2. CONCLUSION: There was a high prevalence of MS in the senile population and MS was associated with higher 10 years mortality of cerebro-cardiovascular diseases.

[Association between fasting plasma glucose and the 5 years outcome post PCI in aged patients with coronary artery disease].

OBJECTIVE: To observe the association between fasting plasma glucose (FPG) and 5 years outcome post PCI in aged patients with coronary artery disease (CAD). METHODS: A total of 269 patients (mean age 63.8 +/- 9.4 years, 236 males) with CAD underwent PCI between January 2000 and December 2001 were followed up and data on angiographic restenosis, the major adverse cardiac events, the cumulative survival rates and the correlated risk factors were collected and analyzed. Patients were divided into 4 groups according to the levels of their FPG at baseline (group 1: FPG < 5.6 mmol/L; group 2: 5.6 mmol/L < or = FPG < 6.1 mmol/L; group 3: 6.1 mmol/L < or = FPG < 7.0 mmol/L; group 4: FPG > or = 7.0 mmol/L). RESULTS: At the end of the 5 years follow-up, the incidences of major adverse cardiac events, target lesion revascularization, recurring angina pectoris and angiographic restenosis of group 2 were significantly higher than those of group 1 (P < 0.05) and similar as those in group 3 (P > 0.05). The cumulative survival rates of cardiovascular events of group 2, group 3 and group 4 were all significantly decreased compared with group 1 (P < 0.05). The logistic regression model analysis showed that FPG was an independent risk factor for angiographic restenosis, incidence of major adverse cardiac events, all-cause mortality and recurring angina pectoris (P < 0.05). CONCLUSION: FPG > or = 5.6 mmol/L and over is associated with increased incidences of major adverse cardiac events in aged patients with CAD who underwent PCI.

[The reproducibility and clinical significance of oral glucose tolerance test for abnormal glucose metabolism].

OBJECTIVES: To examine the reproducibility of oral glucose tolerance test (OGTT) in screening abnormal glucose metabolism in Chinese population. METHODS: Two times of OGTT were performed in 259 Chinese subjects without a history of diabetes (male 152, female 107) over a period of 2-3 weeks. RESULTS: The overall reproducibility was 56.4%. Subjects with impaired glucose regulation (IGR) at the first test showed lower reproducibility (37.5%), as compared with subjects with normal glucose metabolism (91.2%) and newly detected diabetes (63.8%). The reproducibility of OGTT in group with two components of metabolic syndrome is the lowest (P < 0.05). According to the results of the two tests, subjects with one abnormal OGTT had older, higher systolic blood pressure, heart rate and waist-to-hip ratio (WHR) than those with two normal OGTTs and had lower waist circumference, triglyceride and higher high-density lipoprotein cholesterol than those with two abnormal OGTTs. CONCLUSIONS: These findings confirmed the poor reproducibility of OGTT which was lower in subjects with IGR and moderate metabolic abnormality. Furthermore, subjects with one abnormal OGTT, whether reproducible or not, had a higher cardiovascular risk profile, as compared with subjects with normal glucose regulation on two OGTTs. Therefore, OGTT is still essential to the screening of abnormal glucose metabolism.