Diagnostic yield of copy number variation sequencing in fetuses with increased nuchal translucency: a retrospective study.

OBJECTIVE: To assess the efficacy of copy number variation sequencing (CNV-seq) and karyotyping for prenatal detection of chromosomal abnormalities in fetuses with increased nuchal translucency. METHODS: Amniotic fluid samples were extracted from 205 fetuses with increased nuchal translucency (NT ≥ 2.5 mm), diagnosed by ultrasound between gestational ages of 11 and 13 + 6 weeks. Karyotyping and CNV-seq were performed for detecting chromosomal abnormalities. RESULTS: There are 40 fetuses (19.51%) showing increased NT detected with chromosomal abnormalities in karyotyping, and trisomy 21 was found to be the most common abnormalities. There are 50 fetuses (24.39%) identified with chromosomal abnormalities by CNV-seq. The detection of the applied techniques indicated that CNV-seq revealed higher chromosomal aberrations. The risk of chromosomal abnormalities was significantly increased with NT thickening, from 13.64% in the NT group of 2.5-3.4 mm, 38.64% in the NT group of 3.5-4.4 mm, and to 51.72% in the NT group of over 4.5 mm (P < 0.05). The investigated cases with increased NT with presence of soft markers in ultrasound or high risk in non-invasive prenatal testing presented chromosomal abnormalities in higher rates, comparing with those with isolated NT or low risk (P < 0.05). CONCLUSION: The results indicated that the risk of chromosomal abnormalities was associated with the NT thickness, detected by karyotype or CNV-seq. The combination application of two analysis was efficient to reveal the possible genetic defects in prenatal diagnosis. The finding suggested that the detection should be considered with ultrasonographic soft markers, and the NT thickness of 2.5-3.4 mm could be a critical value for detecting chromosomal abnormalities to prevent the occurrence of missed diagnosis.

Glucose potentiates ß-cell function by inducing Tph1 expression in rat islets.

Impaired pancreatic ß-cell function is the primary defect in type 2 diabetes. Glucose is an important regulator of ß-cell growth and function; however, the mechanisms that are involved in the chronic adaptation of ß cells to hyperglycemia remain largely unknown. In the present study, global gene expression patterns revealed that tryptophan hydroxylase 1 (Tph1) was the most profound of genes that are up-regulated in rat islets exposed to high glucose. Calcium and cAMP signals synergistically mediated glucose-stimulated Tph1 transcription in ß cells by activating cAMP-responsive element-binding protein and promoting its binding with a Tph1 promoter. Similar to in vitro results, in vivo infusion of high glucose also strongly induced Tph1 expression and serotonin production in rat islets, along with enhanced islet function. Inhibition or knockdown of Tph1 markedly decreased glucose-potentiated insulin secretion. In contrast, overexpression of Tph1 augmented glucose-stimulated insulin secretion in rat islets by up-regulating the expression of genes that are related to islet function. In addition, the long-acting glucagon-like peptide 1 receptor agonist, exendin-4, stimulated Tph1 expression in a glucose-dependent manner. Knockdown of Tph1 inhibited exendin-4-potentiated insulin secretion in rat islets. These findings suggest that Tph1 mediates the compensation of islet function induced by glucose, and that promoting Tph1 expression in pancreatic ß cells will provide a new strategy for the treatment of type 2 diabetes mellitus.-Zhang, Y., Deng, R., Yang, X., Xu, W., Liu, Y., Li, F., Zhang, J., Tang, H., Ji, X., Bi, Y., Wang, X., Zhou, L., Ning, G. Glucose potentiates ß-cell function by inducing Tph1 expression in rat islets.

Acute exposure of beta-cells to troglitazone decreases insulin hypersecretion via activating AMPK.

BACKGROUND: It has been recognized that insulin hypersecretion can lead to the development of insulin resistance and type 2 diabetes mellitus. There is substantial evidence demonstrating that thiazolidinediones are able to delay and prevent the progression of pancreatic ß-cell dysfunction. However, the mechanism underlying the protective effect of thiazolidinediones on ß-cell function remains elusive. METHODS: We synchronously detected the effects of troglitazone on insulin secretion and AMP-activated protein kinase (AMPK) activity under various conditions in isolated rat islets and MIN6 cells. RESULTS: Long-term exposure to high glucose stimulated insulin hypersecretion and inhibited AMPK activity in rat islets. Troglitazone-suppressed insulin hypersecretion was closely related to the activation of AMPK. This action was most prominent at the moderate concentration of glucose. Glucose-stimulated insulin secretion was decreased by long-term troglitazone treatment, but significantly increased after the drug withdrawal. Compound C, an AMPK inhibitor, reversed troglitazone-suppressed insulin secretion in MIN6 cells and rat islets. Knockdown of AMPKα2 showed a similar result. In MIN6 cells, troglitazone blocked high glucose-closed ATP-sensitive K(+) (KATP) channel and decreased membrane potential, along with increased voltage-dependent potassium channel currents. Troglitazone suppressed intracellular Ca(2+) response to high glucose, which was abolished by treatment with compound C. CONCLUSION: Our results suggest that troglitazone provides ß-cell "a rest" through activating AMPK and inhibiting insulin hypersecretion, and thus restores its response to glucose. GENERAL SIGNIFICANCE: These data support that AMPK activation may be an important mechanism for thiazolidinediones preserving ß-cell function.

Role of hepatic neuregulin 4 in the regulation of gluconeogenesis in mice.

AIMS: Enhanced hepatic gluconeogenesis is an important cause of hyperglycemia in type 2 diabetes. However, the regulatory mechanisms underlying disordered hepatic gluconeogenesis remains largely unclear. In the present study, we investigated the potential role of hepatic neuregulin 4 (Nrg4) in the regulation of gluconeogenesis in mice. MAIN METHODS: Microarray analysis was performed in primary mouse hepatocytes treated with or without 8-Br-cAMP. Primary mouse hepatocytes transfected with Nrg4 overexpressing or shRNA adenovirus were used to detect the expressions of the key gluconeogenic genes and glucose output. Hepatic Nrg4 expression levels were measured in fasted C57/BL6 mice, obese ob/ob mice, diabetic db/db mice and Goto-Kakisaki (GK) rats. Pyruvate tolerance test was performed and gluconeogenic gene expressions were detected 7 days after Nrg4 shRNA adenovirus was injected into male C57BL/6 and db/db mice. KEY FINDINGS: Microarray analysis revealed that Nrg4 expression was significantly induced by 8-Br-cAMP in primary mouse hepatocytes, along with the upregulation of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Adenovirus-mediated overexpression or knockdown of Nrg4 in primary mouse hepatocytes increased or decreased PEPCK and G6Pase expressions as well as hepatic glucose production. Hepatic Nrg4 expression was induced by fasting in normal C57/BL6 mice, and markedly upregulated in obese ob/ob mice, diabetic db/db mice and GK rats. Hepatic Nrg4 knockdown in C57BL/6 and db/db mice improved pyruvate tolerance, with the downregulation of PEPCK, G6Pase, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). SIGNIFICANCE: Hepatic Nrg4 plays a crucial role in the regulation of gluconeogenesis and may be a therapeutic target of type 2 diabetes.

PPP1R3C mediates metformin-inhibited hepatic gluconeogenesis.

BACKGROUND: Metformin has been widely used to alleviate hyperglycemia in patients with type 2 diabetes mainly via suppressing hepatic gluconeogenesis. However, the underlying mechanism remains incompletely clear. Here, we aimed to explore the role of PPP1R3C in metformin-mediated inhibition of hepatic gluconeogenesis. METHODS: The differentially expressed genes in primary mouse hepatocytes incubated with 8-Br-cAMP and metformin were analyzed by microarrays. Hepatic glucose production and gluconeogenic gene expressions were detected after adenovirus-mediated overexpression or silence of PPP1R3C in vitro and in vivo. The phosphorylation level and location of transducer of regulated CREB activity 2 (TORC2) were determined by Western blot and immunofluorescence. RESULTS: Metformin and adenovirus-mediated activation of AMPK suppressed 8-Br-cAMP-stimulated Ppp1r3c mRNA expression in primary mouse hepatocytes. Overexpression of PPP1R3C in primary mouse hepatocytes or the livers of wild-type mice promoted hepatic glucose production and gluconeogenic gene expressions. On the contrary, adenovirus-mediated knockdown of PPP1R3C in primary mouse hepatocytes decreased hepatic gluconeogenesis, with the suppression of cAMP-stimulated gluconeogenic gene expressions and TORC2 dephosphorylation. Notably, Ppp1r3c expression was increased in the liver of db/db mice. After PPP1R3C silence in the livers of wild-type and db/db mice, blood glucose levels and hepatic glucose production were markedly lowered, with decreased expressions of key gluconeogenic enzymes and transcript factors as well as liver glycogen content. CONCLUSION: Metformin-activated AMPK decreases hepatic PPP1R3C expression, leading to the suppression of hepatic gluconeogenesis through blocking cAMP-stimulated TORC2 dephosphorylation. Hepatic specific silence of PPP1R3C provides a promising therapeutic strategy for type 2 diabetes.

Expression profile analysis of long non-coding RNAs involved in the metformin-inhibited gluconeogenesis of primary mouse hepatocytes.

Long non-coding RNAs (lncRNAs) have been demonstrated to regulate metabolic tissue development and function, including adipogenesis, hepatic lipid metabolism, islet function and energy balance. However, the role of lncRNAs in gluconeogenesis remains completely unknown. Metformin reduces glucose output mainly via the inhibition of gluconeogenesis. In the present study, the lncRNA expression profile of primary mouse hepatocytes exposed to cyclic adenosine monophosphate (cAMP), a gluconeogenic stimulus, with or without metformin was analyzed by microarray. Among the 22,016 lncRNAs that were identified, 456 were upregulated and 409 were downregulated by cAMP (fold-change ≥2.0). Furthermore, the cAMP-induced upregulation of 189 lncRNAs and downregulation of 167 lncRNAs was attenuated by metformin. The expression levels of eight lncRNAs were validated by reverse transcription-quantitative polymerase chain reaction, and the results were consistent with those of the microarray analysis. Among them, two lncRNAs NR_027710 and ENSMUST00000138573, were identified to have an association with two protein coding genes, namely peroxisome proliferator-activated receptor-γ coactivator-1α, a critical transcriptional coactivator in gluconeogenesis, and G protein-coupled receptor 155, respectively. The two protein coding genes exhibited similar expression patterns to their associated lncRNAs. The findings of the present study suggest that lncRNAs are potentially involved in the regulation of gluconeogenesis.

Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity.

Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome proliferators-activated receptor γ2 (PPARγ2), and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB) phosphorylation and C/EBPß expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPß expression induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE) stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPß was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPß-triggered transcriptional cascades.

[Detection of human parvovirus B19 nonstructural protein DNA by nested- polymerase chain reaction in pregnant women].

OBJECTIVE: To explore the sensitivity and specificity of the new nested-polymerase chain reaction (nested-PCR) assay in the detection of human parvovirus B19 (HPV B19) nonstructural protein DNA and the applicability of this technique in detecting the gravida infected with parvovirus B19. METHODS: Evaluating the new nested-polymerase chain reaction assay by routine process, and compared with the general nested-PCR in detecting structural protein DNA. Then, the two techniques were used to detect human parvovirus B19 structural protein and nonstructural protein DNA in gravida serum and pregnant tissue of 30 cases. RESULTS: The sensitivity of the new method was 0.005 fg which was similar to the general nested-polymerase chain reaction assay, and the detection results of other viruses and human genome DNA were all negative. The positive rates of structural protein DNA and nonstructural protein DNA in gravida sera were 26.7% and 33.3%, respectively. And the difference was non significant in statistics. At the same time, both of these two positive rates in placenta and villus were 26.7%. CONCLUSIONS: The new nested-polymerase chain reaction assay was a sensitive, specific and convenient method in detecting human parvovirus B19 infection, and the detection of nonstructural protein DNA paves the way for the following study on the disease mechanisms of B19.

Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.

Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and ß-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.