Li-Qiong Xue

Stevioside ameliorates high-fat diet-induced insulin resistance and adipose tissue inflammation by downregulating the NF-κB pathway

Accumulating evidence suggests that adipose tissue is the main source of pro-inflammatory molecules that predispose individuals to insulin resistance. Stevioside (SVS) is a widely used sweetener with multiple beneficial effects for diabetic patients. In this study, we investigated the effect of SVS on insulin resistance and the pro-inflammatory state of adipose tissue in mice fed with a high-fat diet (HFD). Oral administration of SVS for 1month had no effect on body weight, but it significantly improved fasting glucose, basal insulin levels, glucose tolerance and whole body insulin sensitivity. Interestingly, these changes were accompanied with decreased expression levels of several inflammatory cytokines in adipose tissue, including TNF-α, IL6, IL10, IL1β, KC, MIP-1α, CD11b and CD14. Moreover, macrophage infiltration in adipose tissue was remarkably reduced by SVS. Finally, SVS significantly suppressed the nuclear factor-kappa b (NF-κB) signaling pathway in adipose tissue. Collectively, these results suggested that SVS may ameliorate insulin resistance in HFD-fed mice by attenuating adipose tissue inflammation and inhibiting the NF-κB pathway.

A refined study of FCRL genes from a genome-wide association study for Graves' disease.

To pinpoint the exact location of the etiological variant/s present at 1q21.1 harboring FCRL1-5 and CD5L genes, we carried out a refined association study in the entire FCRL region in 1,536 patients with Graves’ disease (GD) and 1,516 sex-matched controls by imputation analysis, logistic regression, and cis-eQTL analysis. Among 516 SNPs with P<0.05 in the initial GWAS scan, the strongest signals associated with GD and correlated to FCRL3 expression were located at a cluster of SNPs including rs7528684 and rs3761959. And the allele-specific effects for rs3761959 and rs7528684 on FCRL3 expression level revealed that the risk alleles A of rs3761959 and C of rs7528684 were correlated with the elevated expression level of FCRL3 whether in PBMCs or its subsets, especially in CD19+ B cells and CD8+ T subsets. Next, the combined analysis with 5,300 GD cases and 4,916 control individuals confirmed FCRL3 was a susceptibility gene of GD in Chinese Han populations, and rs3761959 and rs7528684 met the genome-wide association significance level (Pcombined = 2.27×10−12 and 7.11×10−13, respectively). Moreover, the haplotypes with the risk allele A of rs3761959 and risk allele C of rs7528684 were associated with GD risk. Finally, our epigenetic analysis suggested the disease-associated C allele of rs7528684 increased affinity for NF-KB transcription factor. Above data indicated that FCRL3 gene and its proxy SNP rs7528684 may be involved in the pathogenesis of GD by excessive inhibiting B cell receptor signaling and the impairment of suppressing function of Tregs.

Genetic Heterogeneity of Susceptibility Gene in Different Ethnic Populations: Refining Association Study of PTPN22 for Graves' Disease in a Chinese Han Population

In our previous studies, we presumed subtypes of Graves’ disease (GD) may be caused by different major susceptibility genes or different variants of a single susceptibility gene. However, more evidence is needed to support this hypothesis. Single-nucleotide polymorphism (SNP) rs2476601 in PTPN22 is the susceptibility loci of GD in the European population. However, this polymorphism has not been found in Asian populations. Here, we investigate whether PTPN22 is the susceptibility gene for GD in Chinese population and further determine the susceptibility variant of PTPN22 in GD. We conducted an imputation analysis based on the results of our genome-wide association study (GWAS) in 1,536 GD patients and 1,516 control subjects. Imputation revealed that 255 common SNPs on a linkage disequilibrium (LD) block containing PTPN22 were associated with GD (P<0.05). Nine tagSNPs that captured the 255 common variants were selected to be further genotyped in a large cohort including 4,368 GD patients and 4,350 matched controls. There was no significant difference between the nine tagSNPs (P>0.05) in either the genotype distribution or allelic frequencies between patients and controls in the replication study. Although the combined analysis exhibited a weak association signal (P combined = 0.003263 for rs3811021), the false positive report probability (FPRP) analysis indicated it was most likely a false positive finding. Our study did not support an association of common SNPs in PTPN22 LD block with GD in Chinese Han population. This suggests that GD in different ethnic population is probably caused by distinct susceptibility genes.

Mimecan in pituitary corticotroph cells may regulate ACTH secretion and the HPAA

Mimecan is a protein of unknown function that is expressed in the pituitary tissues of mouse and human. In this study, we observed the function of mimecan on the proopiomelanocortin (POMC) gene in the pituitary and the hypothalamo-pituitary-adrenal axis (HPAA). Incubating pituitary corticotroph AtT-20 cells with recombinant mimecan protein stimulated adrenocorticotrophic hormone (ACTH) secretion without significantly up-regulating POMC gene expression. In addition, pituitary corticotroph AtT-20 cell corticotropin-releasing hormone receptor 1 (CRHR1) gene expression was induced by mimecan. Interestingly, long-term mimecan overexpression in corticotroph cells increased CRHR1 mRNA levels while slightly decreasing POMC mRNA expression and ACTH secretion. Using mimecan knockout mice, we found that, although the serum ACTH concentration was not significantly different between wild type and mimecan knockout mice under basal conditions, the serum ACTH level was relatively lower in mimecan knockout mice after treatment with corticotropin-releasing hormone (CRH). Meanwhile, we observed that POMC and CRHR1 gene expression decreased in primary cultured knockout mouse pituitary cells compared with wild type cells. Taken together, these data suggest that mimecan expressed in pituitary corticotroph cells mainly regulates ACTH secretion in the pituitary and coordinates the HPAA.

Functional study of an aberrant splicing variant of the human luteinizing hormone (LH) receptor.

The luteinizing hormone receptor (LHR) is a member of a subfamily of G protein-coupled receptors that is characterized by its alternative splicing. In a previous study, we identified a splice site mutation of intron 6 (IVS6-3C>A) in a patient suffering from Leydig cell hypoplasia, which leads to aberrant splicing of LHR mRNA. In vitro expression analysis confirmed that this mutation results in the skipping of exon 7 in the mature mRNA of the LHR gene. In this study, we determined the impact of IVS6-3C>A on the RNA secondary structure and function of LHR-Del7. The three-dimensional structure of the leucine-rich repeats in LHR was predicted by molecular modeling. Radioactive ligand-binding assays verified that LHR-Del7 has no binding affinity for hCG. Furthermore, we detected negligible cAMP production in cells transfected with LHR-Del7. Cells co-expressing LHR-WT and LHR-Del7 were able to generate cAMP in response to hCG, but there was no significant difference between cells transfected with LHR-WT/vector and LHR-WT/LHR-Del7, although the variant was able to localize to cell surface, similar to wild-type receptor. These results indicated that LHR-Del7 does not have a dominant negative effect on LHR-WT cell surface expression, and although the pathological splicing variant LHR-Del7 was able to localize to cell membranes it failed to bind hCG and had no effect on wild-type LHR.

Genetic Study of Early-onset Graves’ Disease in the Chinese Han Population

Graves’ disease (GD) is a complex autoimmune disorder in which genetic and environmental factors are both involved in the pathogenesis. Early‐onset patients have a shorter exposure time to environmental factors and are, therefore, good models to help understand the genetic architecture of GD. Based on previous studies of early‐onset GD, 11 single nucleotide polymorphisms (SNPs) and their related SNPs (R2 > .6), SNPs located within a ±1‐Mb region of the FOXP3 gene, and 20 validated GD‐risk SNPs were selected and screened for genotyping in 3735 GD and 4893 control patients to investigate whether early‐onset GD is a subtype of GD with distinct susceptibility genes. Ultimately, we did not confirm the reported genetic markers of early‐onset GD in our Chinese Han population but found that a GD‐risk SNP located in the human leukocyte antigen class I region—rs4947296—was more strongly correlated with early‐onset GD than non‐early‐onset GD. In addition, heterogeneity analysis of GD patients suggests that it may be more reasonable to define early‐onset GD as an onset age ≤20 years.

ITM2A Expands Evidence for Genetic and Environmental Interaction in Graves Disease Pathogenesis

Context: Graves disease (GD) is a common autoimmune disease triggered by genetic predisposition and environmental factors. However, the mechanisms of interaction between genetic and environmental factors contributing to the development of GD remain unknown. Objective: We aimed to identify GD susceptibility variants and genes on Xq21.1 locus and interpret the contribution of interaction between genetic predisposition on Xq21.1 and environmental factors to GD. Design: We performed refining study on Xq21.1 in a 2-stage study and carried out expression quantitative trait locus analysis of the best association signal with GD. Setting and Participants: A total of 4316 GD patients and 4374 sex-matched controls were collected from the Chinese Han population by cooperation with multiple hospitals. Results: We identified that rs3827440 or its linkage single nucleotide polymorphisms (SNPs) were probably the causal variant in the Xq21.1 locus, with the most substantial association with GD in our combined cohorts (P = 2.45 × 10−15). The genotypes of rs3827440 were correlated with the expression of ITM2A in monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Notably, the expression of ITM2A in monocytes after lipopolysaccharide (LPS) and interferon-&ggr; (INF-&ggr;) stimulation showed substantial difference among the volunteers that carried different genotypes of rs3827440 (P = 9.40 × 10−7 and P = 1.26 × 10−5 for 24 hours’ LPS and INF-&ggr; stimulation, respectively). Moreover, ITM2A expression was significantly decreased in PBMCs from untreated GD patients than that from controls. Conclusion: The results suggest that ITM2A might be a susceptibility gene for GD in the Xq21.1 locus, and environmental factors, such as viral and bacterial infections, probably contribute to GD pathogenesis by interacting with the risk SNP rs3827440 mediating the regulation of ITM2A expression.

Identification of a novel mutation in CYP17A1 gene

17α-hydroxylase/17,20-lyase deficiency (17OHD) is a rare autosomal recessive genetic disease that is characterized by low-renin hypertension, hypokalemia, and abnormal development of the genitalia. Mutations in the CYP17A1 gene account for this disease. We aim to investigate the CYP17A1 mutation and analyze its possible influence on phenotype in a Chinese patient with 17OHD. Steroid hormones were assayed. The 8 exons of the CYP17A1 gene were amplified and directly sequenced. Wild-type and mutant CYP17A1 cDNA were cloned into pcDNA3.1 expression vectors and transfected into 293T cells. Finally, 17-hydroxylase and 17,20-lyase activity were detected by using progesterone and 17-hydroxypregnenolone as the substrates. A novel missense mutation c.716 G>A located in exon 4 that changed the amino acid from arginine to glutamine (R239Q) was discovered in the patient. Steric model analysis of CYP17A1 showed that R239Q changed the local structure and the electrostatic potential. Functional study indicated that the R239Q mutant caused the complete loss of both 17α-hydroxylase and 17,20-lyase activities. Our study expanded the CYP17A1 mutation spectrum. With a functional study, we confirmed that the novel mutation caused the complete loss of both 17α-hydroxylase and 17,20-lyase activities.

Treatment of acute multiorgan dysfunction occurring in congenital adrenal hyperplasia

A 23-year-old female was admitted to the hospital with a 2-day history of vomiting, diarrhea, and fever. The patient had a history of clitoral hypertrophy at birth, which was surgically corrected in her childhood. She presented with primary amenorrhea, and her chromosome karyotype was 46, XX. Approximately 12 months before admission, her steroid hormone profile showed elevated levels of testosterone (T 3.31 ng/ml, 0.15–0.51 ng/ml), dehydroepiandrosterone (DHEA 618.9 lg/dl, 19.00–391.00 lg/dl), progesterone (P 9.79 ng/ml, 0.2–0.9 ng/ml), and 17-hydroxyprogesterone (17OHP 10.99 ng/ml, 0.23–1.36 ng/ml), while the levels of follicle-stimulating hormone (FSH 5.83 mIU/ml, 21–104 mIU/ml) and luteinizing hormone (LH 3.58 mIU/ml, 0.9–58.6 mIU/ml) were decreased. A pelvic ultrasound indicated the presence of a uterus and ovaries. The patient was diagnosed with congenital adrenal hyperplasia (CAH) and 21-hydroxylase deficiency (21OHD). Then, she was treated with cortisone acetate (25 mg qd). Her levels of 17OHP (6.83 ng/ml) and T (0.15 ng/ml) decreased 6 months after treatment. Nine months after the treatment, she experienced menstruation and withdrew herself from the drug. At the time of her arrival at our hospital, a physical examination showed that she was hirsute, and her breasts were underdeveloped. Her heart rate was 100 bpm, and her blood pressure was undetectable. Blood tests indicated a WBC of 9 9 10/l (4–9.2 9 10/l), neutrophils 75.9 % (50–70 %), a PLT of 41 9 10/l (85–303 9 10/l), 6.25 mmol/l (3.5–5.1 mmol/l) potassium, and 134 mmol/l (135–147 mmol/l) sodium. She also had hypoglycemia (2.4 mmol/l, 3.9–6.1 mmol/l), and the levels of serum creatinine (Scr) (445 lmol/l, 53–115 lmol/l), amylase (653 U/l, 25–125 U/l), and TnI (2.5 ng/ml, 0–0.04 ng/ml) were increased. A blood gas analysis demonstrated a pH of 7.11 and a BE of -23.7 mmol/l. A stool occult blood test was positive. On the 2nd day after admission, she showed impaired liver function (GPT 4800 U/l, 1–65 U/l and GOT 14 750 U/l, 15–37 U/l) and decreased urine volume in combination with a gradually increased Scr. She was diagnosed with multiple organ dysfunction syndrome (MODS) and adrenal crisis. DNA was extracted from her peripheral leukocytes, and mutations were analyzed by sequencing the whole CYP21A2 gene. Following conventional treatment, she underwent hemodiafiltration (HDF) on D7 and D8. However, the Scr (D9 327 lmol/l; D13 123 lmol/l) and liver function (D6 GPT 533 U/l and GOT 242 U/l; D10 GPT 204 U/l and GOT 42 U/l) continued to decline (Table 1). Because the patient was in shock and had adrenal cortex dysfunction, she was also administered hydrocortisone (D2 to D6, 200 mg/d). The patient gradually recovered and was discharged from the hospital on D14. When the patient arrived at our hospital, she had gastrointestinal disorders, a normal body temperature and shock. Considering her history of CAH, an adrenal crisis was suspected. An infection and cortisone acetate withdrawal might have been the inducing factors. Therefore, it B. Han Y. Lu J. Qiao (&) Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China e-mail: qiaoj2001@126.com