Yen Hao Chen

miRNA-93 Inhibits GLUT4 and Is Overexpressed in Adipose Tissue of Polycystic Ovary Syndrome Patients and Women With Insulin Resistance

Approximately 70% of women with polycystic ovary syndrome (PCOS) have intrinsic insulin resistance (IR) above and beyond that associated with body mass, including dysfunctional glucose metabolism in adipose tissue (AT). In AT, analysis of the IRS/PI3-K/AKT pathway signaling components identified only GLUT4 expression to be significantly lower in PCOS patients and in control subjects with IR. We examined the role of miRNAs, particularly in the regulation of GLUT4, the insulin-sensitive glucose transporter, in the AT of PCOS and matched control subjects. PCOS AT was determined to have a differentially expressed miRNA profile, including upregulated miR-93, -133, and -223. GLUT4 is a highly predicted target for miR-93, while miR-133 and miR-223 have been demonstrated to regulate GLUT4 expression in cardiomyocytes. Expression of miR-93 revealed a strong correlation between the homeostasis model assessment of IR in vivo values and GLUT4 and miR-93 but not miR-133 and -223 expression in human AT. Overexpression of miR-93 resulted in downregulation of GLUT4 gene expression in adipocytes through direct targeting of the GLUT4 3′UTR, while inhibition of miR-93 activity led to increased GLUT4 expression. These results point to a novel mechanism for regulating insulin-stimulated glucose uptake via miR-93 and demonstrate upregulated miR-93 expression in all PCOS, and in non-PCOS women with IR, possibly accounting for the IR of the syndrome. In contrast, miR-133 and miR-223 may have a different, although yet to be defined, role in the IR of PCOS.

Abnormal Expression of Genes Involved in Inflammation, Lipid Metabolism, and Wnt Signaling in the Adipose Tissue of Polycystic Ovary Syndrome

CONTEXT Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. OBJECTIVE Our objective was to compare gene expression pattern in sc abdominal adipose tissue in nonobese PCOS patients vs. body mass index-matched controls. RESEARCH DESIGN AND METHODS Eleven PCOS subjects and 12 controls (body mass index 20-28 kg/m(2)) were recruited. Total RNA was isolated, and gene expression profiling was performed using Affymetrix Human Genome U133 arrays. Differentially expressed genes were classified by gene ontology. Microarray results for selected genes were confirmed by quantitative real-time PCR (RT-qPCR). Frequently sampled iv glucose tolerance tests were used to assess dynamic insulin sensitivity. RESULTS Ninety-six genes were identified with altered expression of at least 2-fold in nonobese PCOS adipose tissues. Inflammatory response genes were significantly down-regulated. RT-qPCR confirmed decreases in expression of IL6 (12.3-fold), CXCL2 (18.3-fold), and SOCS3 (22.6-fold). Lipid metabolism genes associated with insulin resistance were significantly up-regulated, with confirmed increases in DHRS9 (2.5-fold), UCLH1 (2.6-fold), and FADS1 (2.8-fold) expression. Wnt signaling genes (DKK2, JUN, and FOSB) were differentially expressed. RT-qPCR confirmed significant expression changes in DKK2 (1.9-fold increase), JUN (4.1-fold decrease), and FOSB (60-fold decrease). CONCLUSIONS Genes involved in inflammation, lipid metabolism, and Wnt signaling are differentially expressed in nonobese PCOS adipose tissue. Because these genes are known to affect adipogenesis and insulin resistance, we hypothesize that their dysregulation may contribute to the metabolic abnormalities observed in women with PCOS.

Heregulin regulates Prolactinoma Gene Expression

To investigate the role of p185(her2/neu)/ErbB3 signaling in pituitary tumor function, we examined these receptors in human prolactinomas. Immunofluorescent p185(her2/neu) was detected in almost all (seven of eight), and ErbB3 expression in a subset (four of eight) of tumors (seven adenomas and one carcinoma). Quantitative PCR also showed abundant ErbB3 mRNA in tumor specimens derived from a rarely encountered prolactin-cell carcinoma. Activation of p185(c-neu)/ErbB3 signaling with heregulin, the ErbB3 ligand, in rat lacto-somatotroph (GH4C1) tumor cells specifically induced prolactin (PRL) mRNA expression approximately 5-fold and PRL secretion approximately 4-fold, whereas growth hormone expression was unchanged. Heregulin (6 nmol/L) induced tyrosine phosphorylation and ErbB3 and p185(c-neu) heterodimerization, with subsequent activation of intracellular ERK and Akt. The Akt signal was specific to ErbB3 activation by heregulin, and was not observed in response to epidermal growth factor activation of epidermal growth factor receptor. Gefitinib, the tyrosine kinase inhibitor, suppressed heregulin-mediated p185(c-neu)/ErbB3 signaling to PRL. Heregulin induction of PRL was also abrogated by transfecting cells with short interfering RNA directed against ErbB3. Pharmacologic inhibition of heregulin-induced phosphoinositide-3-kinase/Akt (with LY294002) and ERK (with U0126) signaling, as well as short interfering RNA-mediated mitogen-activated protein kinase-1 down-regulation, showed ERK signaling as the primary transducer of heregulin signaling to PRL. These results show ErbB3 expression in human prolactinomas and a novel ErbB3-mediated mechanism for PRL regulation in experimental lactotroph tumors. Targeted inhibition of up-regulated p185(c-neu)/ErbB3 activity could be useful for the treatment of aggressive prolactinomas resistant to conventional therapy.

MicroRNA-223 Expression Is Upregulated in Insulin Resistant Human Adipose Tissue

MicroRNAs (miRNAs) are short noncoding RNAs involved in posttranscriptional regulation of gene expression and influence many cellular functions including glucose and lipid metabolism. We previously reported that adipose tissue (AT) from women with polycystic ovary syndrome (PCOS) or controls with insulin resistance (IR) revealed a differentially expressed microRNA (miRNA) profile, including upregulated miR-93 in PCOS patients and in non-PCOS women with IR. Overexpressed miR-93 directly inhibited glucose transporter isoform 4 (GLUT4) expression, thereby influencing glucose metabolism. We have now studied the role of miR-223, which is also abnormally expressed in the AT of IR subjects. Our data indicates that miR-223 is significantly overexpressed in the AT of IR women, regardless of whether they had PCOS or not. miR-223 expression in AT was positively correlated with HOMA-IR. Unlike what is reported in cardiomyocytes, overexpression of miR-223 in human differentiated adipocytes was associated with a reduction in GLUT4 protein content and insulin-stimulated glucose uptake. In addition, our data suggests miR-223 regulates GLUT4 expression by direct binding to its 3′ untranslated region (3′UTR). In conclusion, in AT miR-223 is an IR-related miRNA that may serve as a potential therapeutic target for the treatment of IR-related disorders.

Fibroblast Growth Factor-2 Autofeedback Regulation in Pituitary Folliculostellate TtT/GF Cells

To investigate paracrine regulation of pituitary cell growth, we tested fibroblast growth factor (FGF) regulation of TtT/GF folliculostellate (FS) cells. FGF-2, and FGF-4 markedly induced cell proliferation, evidenced by induction of pituitary tumor transforming gene-1 (Pttg1) mRNA expression and percentage of cells in S phase. Signaling for FGF-2-induced FS cell proliferation was explored by specific pharmacological inhibition. A potent inhibitory effect on FGF-2 action was observed by blocking of Src tyrosine kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (>or=0.1 microm), followed by protein kinase C (PKC) inhibition with GF109203X. Treatment with FGF-2 (30 ng/ml; 10 min) activated phosphorylation of signal transducer and activator of transcription-3, ERK, stress-activated protein kinase/c-Jun N-terminal kinase, Akt, and focal adhesion kinase. Src inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine suppressed FGF-2-induced Akt and focal adhesion kinase, indicating effects downstream of FGF-2-induced Src activation. FGF-2 also markedly induced its own mRNA expression, peaking at 2-4 h, and this effect was suppressed by Src tyrosine kinase inhibition. The PKC inhibitor GF109203X abolished FGF-2 autoinduction, indicating PKC as the primary pathway involved in FGF-2 autoregulation in these cells. In addition to pituitary FGF-2 paracrine activity on hormonally active cells, these results show an autofeedback mechanism for FGF-2 in non-hormone-secreting pituitary FS cells, inducing cell growth and its own gene expression, and mediated by Src/PKC signaling.

Steroidogenic Regulatory Factor FOS Is Underexpressed in Polycystic Ovary Syndrome (PCOS) Adipose Tissue and Genetically Associated with PCOS Susceptibility

CONTEXT Polycystic ovary syndrome (PCOS) is a heterogeneous common genetic disorder characterized by hyperandrogenemia and insulin resistance. Alterations in gene expression profiles of the ovary and adipose tissue identified the candidate gene FBJ murine osteosarcoma viral oncogene homolog (FOS) for further investigation of expression changes in metabolic tissues and genetic studies. OBJECTIVE The objective of the study was to confirm the underexpression of the FOS gene in sc adipose and determine whether variants in this gene are risk factors for PCOS. DESIGN RT-PCR was performed in sc fat from women with and without PCOS. Genotyping of single-nucleotide polymorphisms in the FOS locus was performed to test for association with PCOS. SETTING The study was conducted at a tertiary care academic institution. PARTICIPANTS Twenty-two PCOS and 13 control subjects were recruited for gene expression studies. We assembled a discovery genotyping cohort of 354 cases and 161 controls and a replication cohort of 476 cases and 315 controls, all of whom were Caucasian. MAIN MEASUREMENTS Gene expression by quantitative real-time RT-PCR, FOS genotype, and PCOS status were measured. RESULTS FOS expression was confirmed to be reduced in PCOS adipose tissue. Three single-nucleotide polymorphisms were significantly associated with PCOS in the discovery cohort (rs8006998, P = 0.0031; rs8013918, P = 0.0006; rs8013942, P = 0.0087). rs8006998 was also associated with PCOS in the replication cohort (P = 0.013). CONCLUSIONS Differential gene expression in sc fat and genetic association at the FOS locus in PCOS subjects implicates a role for this transcription factor in PCOS. FOS dysfunction may be a common factor between hyperandrogenism and insulin resistance.

Berberine inhibits the proliferation of human uterine leiomyoma cells

OBJECTIVE To determine whether berberine (BBR), a naturally occurring plant-derived alkaloid, inhibits the proliferation of human uterine leiomyoma (UtLM) cells. DESIGN Laboratory research. SETTING Laboratory. PATIENT(S) UtLM and normal human uterine smooth muscle (UtSMC) cell lines. INTERVENTION(S) Treatment with [1] BBR (10, 20, and 50 μM), [2] BBR (20 and 50 μM) and/or 17β-estradiol (E2; 10 and 100 nM), and [3] BBR (20 and 50 μM) and/or progesterone (P4; 10 and 100 nM) for 24 or 72 hours. MAIN OUTCOME MEASURE(S) Cell proliferation, cell cycle, apoptosis, and related genes expression were determined. RESULT(S) BBR inhibited UtLM cell proliferation by inducing G2/M cell cycle arrest and apoptosis. Cell cycle G2/M phase-related genes were altered by BBR treatment: the expression of cyclin A1, cyclin B1, and Cdk1 were down-regulated, while Cdk4, p21, and p53 were up-regulated. BBR-treated cells stained positively for annexin V and manifested increased BAX expression. E2- and P4-induced UtLM cell proliferation was blocked by BBR treatment. In marked contrast, even the highest concentration of BBR (50 μM) did not influence cell proliferation in UtSMC cells. CONCLUSION(S) BBR selectively inhibits cellular proliferation and blocks E2- and P4-induced cell proliferation in UtLM but not in normal UtSMC cells. In addition, BBR did not demonstrate cytotoxicity effects in normal human UtSMCs. Our results suggest BBR could be a potential therapeutic agent for the treatment of uterine leiomyoma.

The expression of the miR-25/93/106b family of micro-RNAs in the adipose tissue of women with polycystic ovary syndrome

CONTEXT In adipose tissue (AT) micro-RNA-93 (miR-93) is significantly overexpressed in polycystic ovary syndrome (PCOS) women and non-PCOS women with insulin resistance (IR). Overexpressed miR-93 directly inhibits glucose transporter isoform 4, impairing both glucose metabolism and insulin sensitivity. The mechanisms behind increased miR-93 expression are unclear. OBJECTIVE Our objective was to determine whether miR-93 expression is concordant with its host gene, MCM7, which contains the miR-25/93/106b gene cluster. PATIENTS AT was excised from 16 women with PCOS (eight with and eight without IR) and 15 non-PCOS (nine with and six without IR). MAIN OUTCOME MEASURES Expression of MCM7 and miR-25/93/106b was measured in AT and 3T3-L1 cells. RESULTS MCM7 expression was lower in both non-PCOS/IR and PCOS women and tended to be lowest in women with PCOS and IR. Overall, the expression of MCM7 in human AT was negatively associated with miR-93 expression and with increased subject IR. Additionally, miR-25 and miR-106b expression is uncoupled from the MCM7 host gene and are positively correlated with IR, although no PCOS-specific difference was observed. MCM7 expression appears to be negatively correlated with increasing fasting glucose. In 3T3-L1 adipocytes, increasing glucose had no effect on miR-93 or miR-25, although it reduced MCM7 and increased miR-106b expression in a dose-dependent fashion. In turn, in 3T3-L1 adipocytes, increasing insulin had no effect on either MCM7 or miR-25/93/106b expression. CONCLUSIONS Our data suggest that the expression of MCM7 and miR-93/25 is PCOS and IR related, whereas that of miR-106b is related to IR only. In 3T3-L1 adipocytes, neither hyperglycemia nor hyperinsulinemia altered the expression of miR-93 or miR-25, although increasing glucose levels down-regulated MCM7 and paradoxically increased that of miR-106b expression. The expression of the miR-25/93/106b family may be regulated through mechanisms distinct from its host gene, MCM7. Finally, our studies suggest potential epigenetic mechanisms for both IR and PCOS.

Serum complexed and free prostate-specific antigen (PSA) for the diagnosis of the polycystic ovarian syndrome (PCOS)

Abstract Background: Polycystic ovarian syndrome (PCOS) is a common cause of reproductive and metabolic dysfunction. We hypothesized that serum prostate-specific antigen (PSA) may constitute a new biomarker for hyperandrogenism in PCOS. Methods: We conducted a cross-sectional study of 45 women with PCOS and 40 controls. Serum from these women was analyzed for androgenic steroids and for complexed PSA (cPSA) and free PSA (fPSA) with a novel fifth- generation assay with a sensitivity of ~10 fg/mL for cPSA and 140 fg/mL for fPSA. Results: cPSA and fPSA levels were about three times higher in PCOS compared to controls. However, in PCOS, cPSA and fPSA did not differ according to waist-to-hip ratio, Ferriman-Gallwey score, or degree of hyperandrogenemia or oligo-ovulation. In PCOS and control women, serum cPSA and fPSA levels were highly correlated with each other, and with free and total testosterone levels, but not with other hormones. Adjusting for age, body mass index (BMI) and race, cPSA was significantly associated with PCOS, with an odds ratio (OR) of 5.67 (95% confidence interval [CI]: 1.86, 22.0). The OR of PCOS for fPSA was 7.04 (95% CI: 1.65, 40.4). A multivariate model that included age, BMI, race and cPSA yielded an area-under-the-receiver-operating-characteristic curve of 0.89. Conclusions: Serum cPSA and fPSA are novel biomarkers for hyperandrogenism in PCOS and may have value for disease diagnosis.

Genetic basis of eugonadal and hypogonadal female reproductive disorders

This review discusses the current state of our understanding regarding the genetic basis of the most important reproductive disorders in women. For clarity, these disorders have been divided into eugonadal and hypogonadal types. Hypogonadal disorders have been further subdivided according to serum gonadotropin levels. Our review focuses on historical and recent data regarding the genetics of the hypothalamic-pituitary-gonadal axis dysfunction, as well as the development and etiology of eugonadal disorders including leiomyomata, endometriosis, spontaneous ovarian hyperstimulation syndrome, polycystic ovarian syndrome, mullerian aplasia, and steroid hormone resistance syndromes. We discuss the known genes most commonly involved in hypergonadotropic hypogonadism (Turner syndrome and premature ovarian failure) and hypogonadotrophic hypogonadism (Kallmann syndrome and normosmic types). In addition, we summarize the current clinical testing approaches and their utility in practical application.