Genetic variants in epoxyeicosatrienoic acid processing and degradation pathways are associated with gestational diabetes mellitus.

AIM: To explore the genetic effects of CYP2C8, CYP2C9, CYP2J2, and EPHX2, the key genes involved in epoxyeicosatrienoic acid processing and degradation pathways in gestational diabetes mellitus (GDM) and metabolic traits in Chinese pregnant women. METHODS: A total of 2548 unrelated pregnant women were included, of which 938 had GDM and 1610 were considered as controls. Common variants were genotyped using the Infinium Asian Screening Array. Association studies of single nucleotide polymorphisms (SNPs) with GDM and related traits were performed using logistic regression and multivariable linear regression analyses. A genetic risk score (GRS) model based on 12 independent target SNPs associated with GDM was constructed. Logistic regression was used to estimate odds ratios and 95% confidence intervals, adjusting for potential confounders including age, pre-pregnancy body mass index, history of polycystic ovarian syndrome, history of GDM, and family history of diabetes, with GRS entered both as a continuous variable and categorized groups. The relationship between GRS and quantitative traits was also evaluated. RESULTS: The 12 SNPs in CYP2C8, CYP2C9, CYP2J2, and EPHX2 were significantly associated with GDM after adjusting for covariates (all P < 0.05). The GRS generated from these SNPs significantly correlated with GDM. Furthermore, a significant interaction between CYP2J2 and CYP2C8 in GDM (PInteraction = 0.014, ORInteraction= 0.61, 95%CI 0.41-0.90) was observed. CONCLUSION: We found significant associations between GDM susceptibility and 12 SNPs of the four genes involved in epoxyeicosatrienoic acid processing and degradation pathways in a Chinese population. Subjects with a higher GRS showed higher GDM susceptibility with higher fasting plasma glucose and area under the curve of glucose and poorer ß-cell function.

Common single-nucleotide polymorphisms combined with a genetic risk score provide new insights regarding the etiology of gestational diabetes mellitus.

AIMS: Few studies have constructed a genetic risk score (GRS) to predict the risk of gestaional diabetes mellitus (GDM). We tested the hypothesis that single-nucleotide polymorphisms (SNPs) confirmed for diabetes and obesity and the GRS are associated with GDM. METHODS: We conducted a case-control study comprising 971 GDM cases and 1682 controls from the University of Hong Kong Shenzhen Hospital. A total of 1448 SNPs reported with type 2 diabetes (T2D), type 1 diabetes (T1D), and obesity were selected and the GRS based on SNPs associated with GDM was created. RESULTS: We confirmed that rs10830963 (OR = 1.41,95% CI = 1.25, 1.59) in MTNR1B and rs2206734 (OR = 1.38, 95% CI = 1.22, 1.55) in CDKAL1 were strongly associated with the risk of GDM. Compared with participants with GRS based on T2D SNPs in the low tertile, the ORs of GDM across increasing GRS tertiles were 1.63 (95% CI 1.29, 2.06) and 2.72 (95% CI 2.18, 3.38) in the middle and high tertile, respectively. The positive associations between the GRS and the risk of GDM were also observed in GRS based on obesity/waist-to-hip ratio (WHR)/body mass index (BMI) SNPs. The resulting GRS for each allele increase was significantly associated with higher glycemic indices and lower HOMA-B values for GRS based on T2D SNPs, but not for GRS based on T1D SNPs and GRS based on obesity/WHR/BMI SNPs. CONCLUSION: These findings indicate that GDM may share a common genetic background with T2D and obesity and that SNPs associated with insulin secretion defects have a vital role in the development of GDM.

11ß-HSD1 inhibition reduces atherosclerosis in mice by altering proinflammatory gene expression in the vasculature.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11ß-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11ß-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11ß-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11ß-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11ß-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11ß-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11ß-HSD1 inhibition. Taken together, our data suggest 11ß-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers.

Common inflammatome gene signatures as well as disease-specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co-expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue-specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response-related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non-drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.

The mediating role of maternal metabolites between lipids and adverse pregnancy outcomes of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is one of the most common complications of pregnancy, and the demographics of pregnant women have changed in recent decades. GDM is a metabolic disease with short- and long-term adverse effects on both pregnant women and newborns. The metabolic changes and corresponding risk factors should be of great significance in understanding the pathological mechanism of GDM and reducing the incidence of adverse pregnancy outcomes in patients with GDM. The well-known GDM-associated lipids used in clinical tests, such as triglyceride (TG), are thought to play a major role in metabolic changes during GDM, which have a potential causal relationship with abnormal pregnancy outcomes of GDM. Therefore, this study analyzed the relationship between clinical lipid indicators, metabolic profiles, and abnormal pregnancy outcomes in GDM through mediation analysis. By constructing a metabolic atlas of 399 samples from GDM patients in different trimesters, we efficiently detected the key metabolites of adverse pregnancy outcomes and their mediating roles in bridging abnormal lipids and adverse pregnancy outcomes in patients with GDM. Our study confirmed that TG and total cholesterol were independent risk factors for adverse pregnancy outcomes in patients with GDM. Several key metabolites as mediators (e.g., gamma-linolenic acid, heptadecanoic acid, oleic acid, palmitic acid, and palmitoleic acid) have been identified as potential biomarkers for adverse pregnancy outcomes in patients with GDM. These metabolites mainly participate in the biosynthesis of unsaturated fatty acids, which may shed new light on the pathology of GDM and provide insights for further exploration of the molecular mechanisms underlying adverse pregnancy outcomes.

Discovery of metabolic biomarkers for gestational diabetes mellitus in a Chinese population.

BACKGROUND: Gestational diabetes mellitus (GDM), one of the most common pregnancy complications, can lead to morbidity and mortality in both the mother and the infant. Metabolomics has provided new insights into the pathology of GDM and systemic analysis of GDM with metabolites is required for providing more clues for GDM diagnosis and mechanism research. This study aims to reveal metabolic differences between normal pregnant women and GDM patients in the second- and third-trimester stages and to confirm the clinical relevance of these new findings. METHODS: Metabolites were quantitated with the serum samples of 200 healthy pregnant women and 200 GDM women in the second trimester, 199 normal controls, and 199 GDM patients in the third trimester. Both function and pathway analyses were applied to explore biological roles involved in the two sets of metabolites. Then the trimester stage-specific GDM metabolite biomarkers were identified by combining machine learning approaches, and the logistic regression models were constructed to evaluate predictive efficiency. Finally, the weighted gene co-expression network analysis method was used to further capture the associations between metabolite modules with biomarkers and clinical indices. RESULTS: This study revealed that 57 differentially expressed metabolites (DEMs) were discovered in the second-trimester group, among which the most significant one was 3-methyl-2-oxovaleric acid. Similarly, 72 DEMs were found in the third-trimester group, and the most significant metabolites were ketoleucine and alpha-ketoisovaleric acid. These DEMs were mainly involved in the metabolism pathway of amino acids, fatty acids and bile acids. The logistic regression models for selected metabolite biomarkers achieved the area under the curve values of 0.807 and 0.81 for the second- and third-trimester groups. Furthermore, significant associations were found between DEMs/biomarkers and GDM-related indices. CONCLUSIONS: Metabolic differences between healthy pregnant women and GDM patients were found. Associations between biomarkers and clinical indices were also investigated, which may provide insights into pathology of GDM.

Intrahepatic Cholestasis of Pregnancy and Associated Adverse Maternal and Fetal Outcomes: A Retrospective Case-Control Study.

OBJECTIVE: Intrahepatic cholestasis of pregnancy (ICP) is a common pregnancy-related liver disease and is associated with an increased risk of adverse neonatal outcomes. Ursodeoxycholic acid (UDCA) is the most effective treatment. This study was aimed at investigating the adverse outcomes of ICP and evaluating the effects of treatment with UDCA in patients with ICP. METHODS: We included 114 women with ICP and 3725 women without ICP (no-ICP group) who delivered in our hospital between September 2017 and August 2019. The prevalence of ICP in this study was 3.15%. We matched each woman with ICP to five controls. Of all the 114 women with ICP, 73 (64.04%) received UDCA while 41 (35.96%) did not. Logistic multivariate regression analysis was used to compare the adverse outcomes between those with ICP and matched controls as well as between those who received UDCA (UDCA group) and those who did not (non-UDCA group). RESULTS: Compared with controls, women with ICP were more likely to have preeclampsia (adjusted odds ratio, aOR = 16.74, 95% CI 5.29-52.98), cesarean section (aOR = 1.76, 95% CI 1.10-2.81), and preterm birth (aOR = 24.35, 95% CI 2.74-216.67). Administration of UDCA reduced the rate of preterm birth (1.37% vs. 14.63%, aOR = 0.10, 95% CI 0.01-0.90). CONCLUSION: ICP increased the risk of preeclampsia, cesarean section, and preterm birth. UDCA could reduce the rate of preterm birth.

Nerve growth factor is closely related to glucose metabolism, insulin sensitivity and insulin secretion in the second trimester: a case-control study in Chinese.

OBJECTIVE: Inflammation-related factors have been shown to play a significant role throughout pregnancy. In this study, we aimed to explore the relationships between selected inflammatory cytokines and gestational diabetes (GDM) in Chinese pregnant women. DESIGN AND METHODS: This was a 1:1 matched case-control study that included 200 pairs of subjects in the second trimester and 130 pairs of subjects in the third trimester. Serum levels of nerve growth factor (NGF), Interleukin-6 (IL-6), leptin, Interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α) and Interleukin-1beta (IL-1ß) were measured by enzyme immunoassay. The associations of these inflammatory factors with metabolic parameters were analysed. RESULTS: In the second trimester, GDM patients had higher NGF levels and lower IL-8 levels than did normal controls (P < 0.001 and P = 0.015, respectively). However, in the third trimester, only lower leptin levels were observed in the GDM group (P = 0.031). Additionally, in the second trimester, NGF levels were not only positively associated with fasting, 1-h and 2-h glucose levels and the area under curve of glucose, but also positively related to insulin sensitivity and secretion, as suggested by fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR) and homeostasis model assessment index of ß-cell secretion (HOMA-ß) (all P < 0.05). Moreover, IL-6 and leptin levels were positively correlated with HOMA-IR and HOMA-ß, and TNF-α levels were positively related to HOMA-IR (all P < 0.05). Except for the relationships between NGF and HOMA-ß and TNF-α and HOMA-IR, the other correlations still existed even after adjusting for confounding factors (all P < 0.05). CONCLUSION: In addition to the positive associations of IL-6 and leptin with insulin resistance and secretion, NGF was higher in the GDM patients and strongly linked to glucose metabolism, insulin resistance and pancreatic ß cell function in Chinese pregnant women in the second trimester.

Serum growth differentiation factor 15 is associated with glucose metabolism in the third trimester in Chinese pregnant women.

OBJECTIVE: Growth differentiation factor 15 (GDF15) has been demonstrated to increase in diabetes as a protective factor. However, studies assessing relationships between GDF15 levels and gestational diabetes mellitus (GDM) are limited. In this study, we aimed to investigate whether GDF15 levels are related to GDM in Chinese subjects. METHODS: We included 200 GDM patients and 200 matched normal controls in the second trimester as well as 130 GDM patients and 130 matched normal controls in the third trimester. Serum GDF15 levels of all participants were determined using an enzyme-linked immunosorbent assay (ELISA). Then, according to GDF15 levels, we equally divided the participants in the second and third trimesters into four subgroups respectively. The relationships of serum GDF15 levels with glucolipid metabolism indicators were analyzed. RESULTS: In the third trimester, GDF15 levels were significantly higher in the GDM patients than in the normal controls (P < 0.001). Additionally, fasting blood glucose (FBG), 1-h postprandial glucose (1h-PG), 2-h postprandial glucose (2h-PG), hemoglobin A1C (HbA1c) and area under curve of glucose (AUCG) from the 75-g oral glucose tolerance test (OGTT) were positively associated with GDF15 levels (P < 0.05), even after adjusting for age, pregestational BMI, changes of BMI until the third trimester, gestational age, twin and family history of diabetes. Moreover, GDF15 levels were higher in the third trimester than in the second trimester (P < 0.001). No significant relationships were found between GDF15 levels and glucolipid metabolism in the second trimester (P > 0.05). CONCLUSIONS: Serum GDF15 levels were positively correlated with glucose metabolism in the third trimester in Chinese pregnant women.

[The effect of calorie restriction on the expression of liver's gluconeogenesis genes of rats fed a high fat diet].

OBJECTIVE: To observe the effect of calorie restriction on the high fat diet rats mRNA expressions of liver forkhead box O1(FoxO1), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G-6-P) and to explore the possible mechanisms. METHODS: 24 normal 6-week-old male Wistar rats were randomly divided into three groups: normal chow group (NC, n = 7), high fat diet group (HF, n = 9) and calorie restriction group (CR, n = 8). They were fed for 12 weeks. At the end of the experiment, the rats were sacrificed and their fasting blood glucose (FBG), insulin (INS), triglycerides (TG), total cholesterol (TC) were measured. Their visceral fat (VF) and body weight (BW) were also measured and VF/BW was calculated. Gene expression was investigated by using semi-quantitative RT-PCR methods. Liver histology was studied with HE stained slides. RESULTS: Compared with the NC group, HF group rats developed visceral obesity which was accompanied by higher FBG, plasma INS, TG, and TC. The levels of FoxO1, PEPCK, and G-6-P increased by 18.9%, 33.8%, and 24.6%, respectively (P less than 0.01). Liver steatosis was observed with microscopy. The BW, VF FBG, INS, TG and TC of the CR group rats were lower in comparison to those of the HF group. The levels of FoxO1, PEPCK and G-6-P were lower by 26.6%, 35.0%, 34.3% (P less than 0.01). Meanwhile, liver steatosis was also milder. CONCLUSION: Calorie restriction can inhibit the expressions of FoxO1, PEPCK and G-6-P, strengthen insulin signal conduction, suppress gluconeogenesis and thus regulate glycometabolism.

Studies on the Function of Disulfide Bond Cys(112)-Cys(115) in Arrowhead Proteinase Inhibitors.

Two cysteine residues which compose the disulfide bond Cys(112)-Cys(115) in the arrowhead inhibitor were replaced by Ala and Ser respectively, using site-directed mutagenesis. The mutant has similar inhibitory activities as that of the wild type. The result suggests that the disulfide bond of Cys(112)-Cys(115) in the arrowhead inhibitor is not indispensable to its inhibitory activity.

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice.

Hydrogen sulfide (H(2)S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H(2)S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H(2)S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine ß-synthase (CBS) has been shown to catalyze H(2)S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H(2)S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H(2)S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H(2)S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H(2)S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H(2)S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H(2)S for the treatment of cardiovascular and other diseases.

Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.

Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

Identification of RNA binding proteins by UV cross-linking.

One of the major focuses of modern biology is to understand the dynamics of RNA-protein interactions, including factors that interact with mRNA, rRNA, tRNA, snRNA, hnRNA, siRNA, and viral RNA. Identification the direct interactions between proteins and RNA has greatly advanced our knowledge about the function of RNA-protein machines. UV crosslinking is a straightforward and powerful tool in this endeavor.

Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex.

The TREX (transcription/export) complex couples transcription elongation to the nuclear export of mRNAs. In this article, we show that the poly(A)(+) RNA-binding proteins Gbp2 and Hrb1, which resemble the serine-arginine-rich (SR) family of splicing factors found in higher eukaryotes, are specifically associated with the yeast TREX complex. We also show that Gbp2 and Hrb1 interact with Ctk1, a kinase that phosphorylates the C-terminal domain of RNA polymerase II during transcription elongation. Consistent with these findings, Gbp2 and Hrb1 associate with actively transcribed genes throughout their entire lengths. By using an RNA immunoprecipitation assay, we show that Gbp2 and Hrb1 also are bound to transcripts that are derived from these genes. We conclude that recruitment of the SR-like proteins Gbp2 and Hrb1 to mRNA occurs cotranscriptionally by means of association with the TREX complex and/or Ctk1.

A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay.

In eukaryotes, a surveillance mechanism known as nonsense-mediated decay (NMD) degrades the mRNA when a premature-termination codon (PTC) is present. NMD requires translation to read the frame of the mRNA and detect the PTC. During pre-mRNA splicing, the exon-exon junction complex (EJC) is recruited to a region 20-24 nt upstream of the exon junction on the mature mRNA. The presence of a PTC upstream from the EJC elicits NMD. Eukaryotic initiation factor 4A (eIF4A) III is a nuclear protein that interacts physically or functionally with translation initiation factors eIF4G and eIF4B, respectively, and shares strikingly high identity with the initiation factors eIF4AI/II. Here we show that siRNA against eIF4AIII, but not against eIF4AI/II, inhibits NMD. Moreover, eIF4AIII, but not eIF4AI, is specifically recruited to the EJC during splicing. The observations that eIF4AIII is loaded onto the mRNA during splicing in the nucleus, has properties related to a translation initiation factor, and functions in NMD raises the possibility that eIF4AIII substitutes for eIF4AI/II during NMD.

Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery.

Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus followed by mRNA export to the cytoplasm for translation. Here we report the identification of a protein, Sus1, which functions in both transcription and mRNA export. Sus1 is a nuclear protein with a concentration at the nuclear pores. Biochemical analyses show that Sus1 interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, and with the Sac3-Thp1 complex, which functions in mRNA export with specific nuclear pore proteins at the nuclear basket. DNA macroarray analysis revealed that Sus1 is required for transcription regulation. Moreover, chromatin immunoprecipitation showed that Sus1 is associated with the promoter of a SAGA-dependent gene during transcription activation. Finally, mRNA export is impaired in sus1 mutants. These data provide an unexpected connection between the SAGA histone acetylase complex and the mRNA export machinery.