Sex-specific genetic influence on thyroid-stimulating hormone and free thyroxine levels, and interactions between measurements: KNHANES 2013-2015

BACKGROUND: Although a wide range of genetic influences on thyroid-stimulating hormone (TSH) and free thyroxine (fT4) levels have been reported, sex differences in the genetic influences have not been well described. METHODS: We assessed TSH and fT4 levels in 2,250 subjects without thyroid peroxidase antibody, with data obtained from the Korea National Health and Nutrition Examination Surveys (KNHANES) conducted from 2013 to 2015. Using variance decomposition methods, the variation of TSH and fT4 levels was divided into genetic and environmental components common to both sexes, and to males and females separately. The genetic correlation between TSH and fT4 levels was also assessed in both sexes, and in males and females separately. RESULTS: Narrow-sense heritability for TSH and fT4 were 54% and 56%, respectively. Sex-specific heritability for TSH levels was significantly higher in females than in males (75% and 41%, respectively; p = 0.037). Heritability for fT4 levels was not significantly different between males and females (62% and 52%, respectively; p = 0.335). TSH and fT4 levels showed a negative genetic correlation in females (ρg = -0.347, p = 0.040) after regressing out the influences of environmental covariates, but this correlation was not present in males (ρg = -0.160, p = 0.391). CONCLUSIONS: The genetic influences on individual TSH levels were more prominent in females than in males. In addition, female-specific pleiotropy between TSH and fT4 might be a clue that this stronger genetic influences in females would mainly affect thyroid function per se, rather than other TSH-related factors that do not primarily trigger the negative feedback loop between TSH and fT4.ope

Association of fibroblast growth factor 10 with the fibrotic and inflammatory pathogenesis of Graves' orbitopathy

Purpose: The role of fibroblast growth factor (FGF) in orbital fibroblasts (OFs) is rarely known. In this study, we investigated the effect of FGF10 on fibrosis and the inflammation mechanism of Graves' orbitopathy (GO). Methods: Orbital tissue from GO (n = 15) and non-GO (n = 15) was obtained for this study. The mRNA and protein expression levels of FGF10 and FGF receptor 2b (FGFR2b) in orbital tissue were determined by real-time polymerase chain reaction, western blot analysis, and confocal microscopy. The effects of FGF10 on transforming growth factor (TGF)-β1 induced fibrotic proteins and interleukin (IL)-1β- or tumor necrosis factor (TNF)-α- induced inflammatory proteins were investigated using recombinant human (rh) FGF10 and small interfering (si) RNA transfection against FGF10. Results: FGF10 and FGFR2b mRNA expression levels were significantly lower in GO orbital tissues than in non-GO orbital tissues (p = 0.009 and 0.005, respectively). Immunostaining of FGF10 in orbital adipose tissues showed differences in FGF10 expression between GO and control samples. Immunostaining of FGF10 was very weak in the orbital tissues of GO patients. TGF-β1-induced fibronectin, collagen Iα, α-smooth muscle actin protein expression in GO OFs was attenuated by rhFGF10 treatment and increased by knockdown of FGF10 via siFGF10 transfection. Similarly, IL-1β- or TNF-α-induced IL-6, IL-8, and cyclooxygenase-2 protein production in GO OFs was either blocked by rhFGF10 treatment or further upregulated by inhibition of FGF10 via siFGF10 transfection. Conclusions: Our data demonstrate that FGF10 has beneficial effects on the inflammatory and fibrotic mechanisms of GO in primary cultured OFs, providing new insights into GO pathology and the discovery of FGF10 as a promising novel therapeutic application for the treatment of GO.ope

Serum Chemerin Levels are Associated with Visceral Adiposity, Independent of Waist Circumference, in Newly Diagnosed Type 2 Diabetic Subjects

PURPOSE: Chemerin has been suggested to be linked to insulin resistance and type 2 diabetes mellitus (T2DM). However, the relationship between visceral adiposity and chemerin levels remains unclear in subjects with T2DM. In this study, we investigated the relationship between serum chemerin levels and visceral adiposity. MATERIALS AND METHODS: This study included 102 subjects newly diagnosed with T2DM. The relationships between serum chemerin levels and clinical and biochemical parameters were examined. Multiple linear regression analysis was performed to determine the predictable factors of serum chemerin levels. RESULTS: Serum chemerin levels showed significant positive correlations with body mass index (BMI), waist circumference (WC), visceral fat thickness (VFT), insulin levels, the homeostasis model assessment of insulin resistance, and levels of triglycerides (log-transformed) and high-sensitivity C-reactive protein, while showing significant negative correlations with high-density lipoprotein cholesterol. After adjusting for BMI and WC, VFT showed a significant relationship with serum chemerin levels (r=0.222, p=0.027). Moreover, VFT was an independent predictive factor of serum chemerin levels (β=0.242, p=0.041). CONCLUSION: We demonstrated that chemerin is linked to metabolic syndrome components. Moreover, serum chemerin levels were associated significantly with obesity, especially visceral adipose tissue, in subjects with T2DM.ope

Role of the phosphatidylinositol-3-kinase and extracellular regulated kinase pathways in the induction of hypoxia-inducible factor (HIF)-1 activity and the HIF-1 target vascular endothelial growth factor in ovarian granulosa cells in response to follicle-stimulating hormone

FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1alpha protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor. We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1alpha protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin-stimulated translation. FSH increases phosphorylation of the AKT target mouse double-minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1alpha as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1alpha protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-I is sufficient to induce HIF-1alpha protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated ERK kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as vascular endothelial growth factor requires not only PI3-kinase/AKT-mediated activation of mammalian target of rapamycin as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.ope

Inhibitory effects of antagonistic analogs of GHRH on GH3 pituitary cells overexpressing the human GHRH receptor

GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.ope

Optimal Waist Circumference Cutoff Value Based on Insulin Resistance and Visceral Obesity in Koreans with Type 2 Diabetes

BACKGROUND: Visceral obesity is the most powerful contributor to the development of metabolic syndrome (MetS) and cardiovascular diseases. In light of visceral obesity, however, there is a paucity of data on the appropriate cutoff point of waist circumference (WC) in subjects with type 2 diabetes. The aim of this study was to investigate the optimal cutoff value for WC that signals insulin resistance (IR) and visceral obesity in Koreans with type 2 diabetes. METHODS: We evaluated 4,252 patients with type 2 diabetes (male 2,220, female 2,032, mean age 57.24 years) who visited our clinic between January 2003 and June 2009. WC was measured at the midpoint between the lower rib and the iliac crest, and insulin sensitivity was assessed by the rate constant of plasma glucose disappearance (Kitt %/min) using an insulin tolerance test. Visceral fat thickness was measured using ultrasonography. Statistical analysis was performed using receiver operating characteristic curve. RESULTS: The optimal cutoff points for WC for identifying the presence of IR and visceral obesity, as well as two or more metabolic components, were 87 cm for men and 81 cm for women. Moreover, these cutoff points had the highest predictive powers for the presence of visceral obesity. The MetS defined by new criteria correlated with the increased carotid intima-media thickness in female subjects. CONCLUSION: Our results suggest that the optimal cutoff values for WC in Koreans with type 2 diabetes should be reestablished based on IR and visceral obesity.ope

Adenovirus-directed expression of dominant negative estrogen receptor induces apoptosis in breast cancer cells and regression of tumors in nude mice.

BACKGROUND: Estrogen receptors (ER) are expressed in about two thirds of human breast cancer, and are an important pharmacological target for treatment of these tumors. Dominant negative forms of the ER have been suggested as an alternative method to disrupt ER function. In this study, we examined the effect of dominant negative ER mutants (ER1-536 and L540Q) on ER-positive breast cancer cells in vitro and in vivo. MATERIALS AND METHODS: ER-positive T47D breast cancer cells were infected with adenoviral vectors expressing ER1-536 and L540Q to examine the effects of the mutants on gene expression and cell growth. Adenoviral vectors containing the wild type ER (AdwtER) and beta-galactosidase gene (AdGal) were used as controls. RESULTS: Ad1-536 or AdL540Q infection inhibited T47D cell growth and induced apoptosis, increasing Bax protein and phosphorylation of p38 mitogen-activated-protein kinase (MAPK). Consistent with the apoptotic effects in vitro, pre-infection of T47D cells with Ad1-536 or AdL540Q inhibited tumor formation when these cells were introduced into nude mice. In addition, injection of Ad1-536 and AdL540Q into pre-established T47D tumors induced tumor regression. Apoptosis, in conjunction with the activation of caspase-3 and phosphorylation of p38 MAPK, was detected in the shrinking tumors. Overexpression of wild-type ER by AdwtER infection also produced antiproliferative and apoptotic effects, but to a lesser extent than the ER1-536 and L540Q mutants. CONCLUSIONS: These results indicate that dominant negative ER mutants have the potential to induce apoptosis of T47D cells and regression of tumors. The delivery of dominant negative ERs by adenoviral vectors may provide a useful tool for targeted therapy of ER-positive breast cancer.ope

Reappraisal of waist circumference cutoff value according to general obesity

BACKGROUND: Current criterion of waist circumference (WC) for abdominal obesity is not enough to demonstrate characteristics of obese and non-obese populations defined by BMI. The aim of this study was to redefine the cutoff values of WC according to general obesity (BMI ≥ 25 kg/m(2)). METHODS: The receiver operating characteristic curve analysis was performed to determine cutoff values of WC for predicting atherosclerosis according to BMI in 1,063 non-diabetic subjects. To validate this new criterion, diabetic patients (n = 3,690) were divided into three groups based on the current (WC of 90/80 cm for men/women) and new cutoff values of WC: 1) group with WC below the lowest value of two criteria; 2) intermediate group defined as having a WC between them; and 3) group with WC more than the highest value of them. RESULTS: The new cutoff values of WC for predicting atherosclerosis in non-diabetic subjects were 84/76 cm for non-obese men/women, and 93/87 cm for obese men/women, respectively. Of non-obese diabetic patients, the intermediate group (WC 84 ~ 90/76 ~ 80 cm for men/women) was more insulin resistant and showed elevated odds ratio (OR) for having 2 or more metabolic risk factors compared to group with WC below 84/76 cm for men/women [OR 2.48 (95 % CI 1.89-3.25) in men, 2.01 (95% CI 1.45-2.78) in women]. In contrast, among obese diabetic patients, insulin resistance and the likelihood of having 2 or more metabolic risk factors were not different from the intermediate group (WC 90 ~ 93/80 ~ 87 cm for men/women) and group with WC below 90/80 cm for men/women. CONCLUSIONS: The current universal cutoff values of WC may under- or over-estimate the metabolic risks of intermediate groups. Therefore, the WC criteria for abdominal obesity should be applied differently depending on the BMI.ope

Adiponectin stimulates osteoblast differentiation through induction of COX2 in mesenchymal progenitor cells

In bone marrow, osteoblasts and adipocytes are differentiated from mesenchymal progenitor cells and their differentiation is reciprocally regulated by largely unknown mechanisms. In this study, we investigated downstream signaling cascades of adiponectin, a member of the adipocytokine family, in the regulation of osteoblast differentiation. Adiponectin augmented expression of several osteogenic marker genes and increased osteoblast differentiation in mesenchymal progenitor cells. The expression of cyclooxygenase-2 (COX2) was potently increased by adiponectin, whereas inhibition of COX2 activity abolished the effect of adiponectin on osteogenesis. In addition, adiponectin rapidly stimulated p38 mitogen-activated protein kinase via the adiponectin receptor, AdipoR1, which resulted in c-Jun activation for COX2 expression. Adiponectin also stimulated BMP2 expression in a COX2-dependent manner. Moreover, Runx2, a key osteogenic transcription factor, contributed to the acceleration of osteogenesis in the presence of adiponectin. Collectively, the finding that adiponectin could promote osteogenesis through an intracellular signaling cascade in mesenchymal progenitor cells suggests that adiponectin would be a potential therapeutic target for bone-related diseases.ope

Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli

Cre recombinase is widely used to manipulate DNA sequences for both in vitro and in vivo research. Attachment of a trans-activator of transcription (TAT) sequence to Cre allows TAT-Cre to penetrate the cell membrane, and the addition of a nuclear localization signal (NLS) helps the enzyme to translocate into the nucleus. Since the yield of recombinant TAT-Cre is limited by formation of inclusion bodies, we hypothesized that the positively charged arginine-rich TAT sequence causes the inclusion body formation, whereas its neutralization by the addition of a negatively charged sequence improves solubility of the protein. To prove this, we neutralized the positively charged TAT sequence by proximally attaching a negatively charged poly-glutamate (E12) sequence. We found that the E12 tag improved the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) compared with those of TAT-NLS-Cre (TAT-Cre) when expressed in E. coli. Furthermore, the growth of cells expressing E12-TAT-Cre was increased compared with that of the cells expressing TAT-Cre. Efficacy of the purified TAT-Cre was confirmed by a recombination test on a floxed plasmid in a cell-free system and 293 FT cells. Taken together, our results suggest that attachment of the E12 sequence to TAT-Cre improves its solubility during expression in E. coli (possibly by neutralizing the ionic-charge effects of the TAT sequence) and consequently increases the yield. This method can be applied to the production of transducible proteins for research and therapeutic purposes.ope