bluuebunny/crossref_metadata_2025 huggingface dataset:
Ovarian Hyporesponsiveness to Follicle Stimulating Hormone in Adolescent Girls Born Small for Gestational Age
Authors: Ibáñez Lourdes , Potau Neus , Zegher Francis de
Published: January 2014

Girls with reduced prenatal growth are known to have, at birth, a small ovarian fraction of primordial follicles and, in adolescence, a uterus and ovaries of small size. We have now examined whether reduced prenatal growth is also followed by changes in the relationships among FSH, inhibin B and estradiol in adolescent girls. We studied 48 post-menarcheal girls (age 13.6 ± 1.4 yr) who were either born with an appropriate weight for gestational age (AGA; n = 33; mean weight 3.3 Kg) or born small for gestational age (SGA; n = 15; mean weight 2.4 Kg). Serum FSH, inhibin B and estradiol concentrations were measured in the early follicular phase (range: day 5 ± 3). SGA girls had, compared to AGA girls, elevated serum FSH (7.2 ± 0.7 vs 4.5 ± 0.3 IU/mL; p = 0.0002), similar inhibin B (62.1 ± 8.1 vs 60.7 ± 6.5 pg/mL) and lower estradiol concentrations (12.1 ± 1.5 vs 21.2 ± 2.4 pg/mL; p = 0.02). SGA girls thus displayed, early after menarche, a pattern that points to hyporesponsiveness of the ovarian granulosa cell fraction and that is reminiscent of reproductive aging. In conclusion, the gynecological correlates of prenatal growth restriction are herewith extended to include ovarian hyporesponsiveness to FSH in adolescence.
Progesterone Withdrawal Up-Regulates Vascular Endothelial Growth Factor Receptor Type 2 in the Superficial Zone Stroma of the Human and Macaque Endometrium: Potential Relevance to Menstruation*
Authors: Nayak Nihar R. , Critchley Hilary O. D. , Slayden Ov D. , Menrad Andreas , Chwalisz Kristof , Baird David T. , Brenner Robert M.
Published: January 2014

Several reports indicate that vascular endothelial growth factor (VEGF) expression is increased in endometrial glands and stroma during the menstrual phase in the human endometrium. Here we report that VEGF receptor type 2 (KDR), normally expressed only in the vascular endothelium, was dramatically up-regulated in the stromal cells of the superficial endometrial zones during the premenstrual phase in both human and macaque endometrium. This increase was detectable by Northern analysis, in situ hybridization, and immunocytochemistry and was cell specific, zone specific, cycle phase specific, and VEGF receptor type specific. That is, it only occurred during the premenstrual/menstrual phase, did not occur in glandular epithelium, endothelium, or stromal cells of the deepest endometrial zones, and was not observed for VEGF receptor type 1. The up-regulation of stromal KDR was induced by progesterone (P) withdrawal in both women and macaques, and adding back P 24 h after P withdrawal in macaques blocked stromal, but not vascular, endothelial KDR expression. Promatrix metalloproteinase-1 (MMP-1) was coordinately up-regulated in the same stromal cell population by P withdrawal. Because of reports that VEGF can enhance MMP expression, we hypothesize that VEGF-KDR interactions may influence MMP expression in the superficial zones of the primate endometrium during the premenstrual phase, and that these interactions play a role in the induction of menstruation.
Effects of Soy Isoflavones on Markers of Bone Turnover in Premenopausal and Postmenopausal Women*
Authors: Wangen Kerry E. , Duncan Alison M. , Merz-Demlow Barb E. , Xu Xia , Marcus Robert , Phipps William R. , Kurzer Mindy S.
Published: January 2014

Soy isoflavones are hypothesized to exert hormonal effects in women and thus may play a role in bone metabolism throughout life. In 2 randomized, cross-over studies, 14 pre- and 17 postmenopausal women were given 3 soy protein isolates containing different amounts of isoflavones [control, 0.13; low isoflavone (low-iso), 1.00; and high-iso, 2.01 mg/kg body wt·day, averaging 8, 65, and 130 mg/day, respectively], for over 3 months each. Food records, blood samples, and 24-h urine collections were obtained throughout the studies. The endpoints evaluated included plasma or serum concentrations of bone-specific alkaline phosphatase, osteocalcin, insulin-like growth factor-I (IGFI), IGF binding protein-3 (IGFBP3), and urine concentrations of deoxypyridinoline cross-links and carboxy-terminal telopeptide of type I collagen. In premenopausal women, IGFI and IGFBP3 concentrations were increased by the low-iso diet, and deoxypyridinoline cross-links was increased by both the low- and high-iso diets during certain phases of the menstrual cycle. In postmenopausal women, bone-specific alkaline phosphatase was decreased by both the low- and high-iso diets, and there were trends toward decreased osteocalcin, IGFI, and IGFBP3 concentrations with increasing isoflavone consumption. Although soy isoflavones do affect markers of bone turnover, the changes observed were of small magnitude and not likely to be clinically relevant. These data do not support the hypothesis that dietary isoflavones per se exert beneficial effects on bone turnover in women.
Codon-54 Polymorphism of the Fatty Acid-Binding Protein 2 Gene Is Associated with Elevation of Fasting and Postprandial Triglyceride in Type 2 Diabetes*
Authors: Georgopoulos Angeliki , Aras Omer , Tsai Michael Y.
Published: January 2014

Patients with type 2 diabetes are frequently dyslipidemic or hypertriglyceridemic. To assess whether increased intestinal triglyceride input leads to elevated fasting and postprandial triglycerides in type 2 diabetes, we used the codon 54 polymorphism of the fatty acid-binding protein 2 gene, which results in the substitution of threonine (Thr) for alanine and is associated with increased intestinal input of triglyceride. Of the 287 diabetic patients screened, 108 (37.6%) were heterozygous and 31 (10.8%) were homozygous for the Thr-54 allele. Mean (±sem) fasting plasma triglyceride levels in patients with the wild-type (n = 80), those heterozygous for the Thr-54 allele (n = 57), and those homozygous for it (n = 18) were 2.0 ± 0.09, 2.7 ± 0.20, and 3.8 ± 0.43 mmol/L, respectively. A linear relationship of mean fasting plasma triglyceride levels (r2 = 0.97) between the 3 groups was found. After fat ingestion, the postprandial area under the curve of plasma triglyceride (P = 0.025) and chylomicrons (Sf > 400, P = 0.013) was higher in the Thr-54/Thr-54 (n = 6) than in the wild-type (n= 9). Our results are consistent with the hypothesis that, in type 2 diabetes, increased intestinal input of triglyceride can lead to elevated fasting and postprandial plasma triglycerides.
Evidence That Parenteral Testosterone Therapy May Improve Endothelium-Dependent and -Independent Vasodilation in Postmenopausal Women Already Receiving Estrogen
Authors: Worboys Samantha , Kotsopoulos Dimitra , Teede Helena , McGrath Barry , Davis Susan R.
Published: January 2014

The gender difference in cardiovascular disease has been partly attributed to higher androgenic hormone levels. Although testosterone in women may not affect lipids, it remains unknown whether it negates favorable estrogenic effects on endothelial function. We have investigated the effects of testosterone implant therapy on arterial reactivity encompassing endothelial-dependent and -independent vasodilation in women using hormone replacement therapy (HRT). B-mode ultrasound measurements of resting brachial artery diameter, following reactive hyperemia [endothelium-dependent flow- mediated dilation (FMD)] and following glyceryl trinitrate (GTN) (endothelium-independent dilation), were recorded in 33 postmenopausal women stabilized on HRT (>6 months), at baseline, and 6 weeks after a testosterone implant (50 mg), with 15 postmenopausal nonusers of HRT serving as controls. In the brachial artery, baseline resting diameter was similar (0.40 ± 0.01 vs. 0.41 ± 0.01 cm, P = 0.5). In the treated group, testosterone levels increased (0.99 ± 0.08 to 4.99 ± 0.3 nmol/L, P < 0.001), associated with a mean 42% increase in FMD (6.4% ± 0.7 to 9.1% ± 1.1, P = 0.03). The control group did not change (8.1% ± 1.4 to 5.6% ± 1.0, P = 0.4). ANOVA of repeated measures (P = 0.04) and mean change (P = 0.02) in FMD both demonstrated significantly greater improvement with testosterone compared with controls. GTN induced vasodilation increased with testosterone treatment (14.9% ± 0.9 to 17.8% ± 1.2, P = 0.03). Our preliminary data indicate that parenteral testosterone therapy improves both endothelial-dependent (flow-mediated) and endothelium-independent (GTN-mediated) brachial artery vasodilation in postmenopausal women using long-term estrogen therapy. The mechanisms underlying these potentially beneficial cardiovascular effects require further investigation.
Effects of Octreotide Treatment on the Proliferation and Apoptotic Index of GH-Secreting Pituitary Adenomas
Authors: Losa Marco , Ciccarelli Enrica , Mortini Pietro , Barzaghi Raffaella , Gaia Daniela , Faccani Giuliano , Papotti Mauro , Mangili Francesca , Terreni Maria Rosa , Camanni Franco , Giovanelli Massimo
Published: January 2014

To investigate the effects of octreotide administration on the growth rate of GH-secreting pituitary adenomas, we measured both the Ki-67 labeling index (LI) and the apoptotic index in tumor specimens from octreotide-treated or matched untreated acromegalic patients. Thirty-nine patients who received octreotide until the day of or the day before surgery and 39 untreated patients matched for sex, age, tumor size, extension, and invasiveness were studied. Immunocytochemical analysis was performed on paraffin-embedded material using a monoclonal antibody (MIB-1) directed against a proliferation-associated nuclear antigen, Ki-67, to measure the growth fraction. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick endlabeling method, using a monoclonal antibody recognizing areas of DNA fragmentation. The Ki-67 LI and apoptosis were counted on separate slides in at least 1000 evaluable cells.

Octreotide-treated patients showed a lower Ki-67 LI (1.8 ± 0.3%) than untreated controls (3.8 ± 0.7%; P < 0.02). Overall, the mean Ki-67 LI of treated patients was 53% lower than that in untreated patients. The antiproliferative effect of octreotide occurred independently of tumor extension and invasiveness. Octreotide-treated and untreated patients showed similar apoptotic indexes (0.6 ± 0.2% and 0.8 ± 0.3%, respectively). There was a positive correlation between the Ki-67 LI and the apoptotic index (r = 0.29; P< 0.03).

Our study demonstrates that acromegalic patients receiving chronic octreotide treatment have a lower value of the proliferation marker Ki-67, but no significant difference in the apoptotic index compared with matched untreated patients. The antiproliferative effect of octreotide on GH-secreting adenomas should imply a lower risk of tumor growth during long-term chronic treatment with the drug.
Variable Expression of the Transcription Factors cAMP Response Element-Binding Protein and Inducible cAMP Early Repressor in the Normal Adrenal Cortex and in Adrenocortical Adenomas and Carcinomas
Authors: Peri Alessandro , Luciani Paola , Conforti Barbara , Baglioni-Peri Silvana , Cioppi Federica , Crescioli Clara , Ferruzzi Pietro , Gelmini Stefania , Arnaldi Giorgio , Nesi Gabriella , Serio Mario , Mantero Franco , Mannelli Massimo
Published: January 2014

The molecular mechanisms leading to adrenocortical tumorigenesis have been only partially elucidated so far. Because the pituitary hormone ACTH, via activation of the cAMP pathway, regulates both cell proliferation/differentiation and steroid synthesis in the adrenal cortex, in this study we focused on the cAMP-dependent transcription factors cAMP responsive element modulator (CREM) and cAMP responsive element binding protein (CREB). We studied CREM and CREB expression by RT-PCR in human normal adrenal cortex (n = 3), adrenocortical adenomas (n = 8), and carcinomas (n = 8). We found transcripts corresponding to the isoforms α, β, γ, and τ2 of the CREM gene in all of the normal adrenal tissues, in the adenomas, and in seven of eight carcinomas. On the other hand, mRNA for the inducible cAMP early repressor isoforms, which derive from an internal promoter of CREM gene, was detected in the normal adrenal and in seven of eight adenomas, but in only three of eight carcinomas. Similarly, CREB transcripts were readily detectable in all normal adrenals and adenomas, whereas they were not found in four of eight adrenal carcinomas. To further characterize the carcinomas, telomerase activity and the expression of the ACTH receptor gene were determined. Telomerase activity in the carcinomas resulted in levels significantly higher than in the adenomas, whereas the levels of ACTH receptor mRNA were lower in the carcinomas. No correlation was found in the carcinomas between the levels of the ACTH receptor transcript and the loss of expression of CREB/inducible cAMP early repressor, suggesting that this alteration is not secondary to an upstream disregulation at the receptor level. In conclusion, our results suggest that an alteration in cAMP signaling may be associated with malignancies of the adrenal cortex.
Phenotypes Associated with SHOX Deficiency
Authors: Ross Judith L. , Scott Charles , Marttila Pia , Kowal Karen , Nass Andrea , Papenhausen Peter , Abboudi Jack , Osterman Lee , Kushner Harvey , Carter Peter , Ezaki Marybeth , Elder Frederick , Wei Fanglin , Chen Huaqun , Zinn Andrew R.
Published: January 2014

Leri-Weill dyschondrosteosis (LWD) (MIM 127300) is a dominantly inherited skeletal dysplasia characterized phenotypically by Madelung wrist deformity, mesomelia, and short stature. LWD can now be defined genetically by haploinsufficiency of the SHOX (short stature homeobox-containing) gene. We have studied 21 LWD families (43 affected LWD subjects, including 32 females and 11 males, ages 3–56 yr) with confirmed SHOX abnormalities. We investigated the relationship between SHOX mutations, height deficit, and Madelung deformity to determine the contribution of SHOX haploinsufficiency to the LWD and Turner syndrome (TS) phenotypes. Also, we examined the effects of age, gender, and female puberty (estrogen) on the LWD phenotype.

SHOX deletions were present in affected individuals from 17 families (81%), and point mutations were detected in 4 families (19%). In the LWD subjects, height deficits ranged from −4.6 to +0.6 sd (mean ± sd = −2.2 ± 1.0). There were no statistically significant effects of age, gender, pubertal status, or parental origin of SHOX mutations on height z-score. The height deficit in LWD is approximately two thirds that of TS. Madelung deformity was present in 74% of LWD children and adults and was more frequent and severe in females than males. The prevalence of the Madelung deformity was higher in the LWD vs. a TS population. The prevalence of increased carrying angle, high arched palate, and scoliosis was similar in the two populations.

In conclusion, SHOX deletions or mutations accounted for all of our LWD cases. SHOX haploinsufficiency accounts for most, but not all, of the TS height deficit. The LWD phenotype shows some gender- and age-related differences.
Regulation of Interleukin 8 Production and Gene Expression in Human Adipose Tissue in Vitro1
Authors: Bruun Jens M. , Pedersen Steen B. , Richelsen Bjørn
Published: January 2014

A variety of cytokines and other compounds are produced in the human adipose tissue and may have autocrine functions in the adipose tissue as well as be involved in the complications seen in association with obesity. Because it recently has been reported that interleukin 8 (IL-8), through its effects on the macrophage and endothelial cell, may be involved in the pathogenesis of atherosclerosis, we found it of interest to investigate whether IL-8 is produced in human adipose tissue in vitro. Human sc adipose tissue was investigated both in incubations with whole adipose tissue fragments as well as with isolated mature adipocytes. In adipose tissue fragments, IL-1β (3 nm) and tumor necrosis factor α (0.6 nm) were able to stimulate IL-8 production by 12-fold and 5-fold, respectively (P < 0.001), when incubated for 48 h. Incubations with isolated adipocytes were performed up to 6 h, and IL-1β and tumor necrosis factor α significantly increased IL-8 production by 50–60% (P < 0.05). Dexamethasone (50 nm) decreased IL-8 production from adipose tissue fragments by 57% (P < 0.01) and from adipocytes by 37% (P < 0.05). IL-8 messenger RNA expression in adipocytes incubated with IL-1β was increased already after 2 h (P < 0.05). Thus, the effect of proinflammatory cytokines and dexamethasone on IL-8 production in adipose tissue seems to be mediated at the transcriptional level. In conclusion, it is demonstrated for the first time that IL-8 is produced and released from human adipose tissue and from isolated adipocytes in vitro, which may indicate that IL-8 from adipose tissue could be involved in some of the obesity-related complications.
A Prospective, Randomized Study of Endometrial Telomerase during the Menstrual Cycle
Authors: Williams Christopher D. , Boggess John F. , LaMarque L. Robert , Meyer William R. , Murray Michael J. , Fritz Marc A. , Lessey Bruce A.
Published: January 2014

The purpose of this study was to characterize telomerase activity during the menstrual cycle, focusing on the luteal phase. A total of 84 endometrial biopsy samples were obtained from 72 participants. Daily urinary LH testing (OvuQuick, Quidel) was used to establish the day of the LH rise, and participants were randomized to return during the secretory phase. Twelve women returned on the identical day during the luteal phase of a subsequent cycle to allow intercycle comparisons of telomerase activity. Telomerase activity was evaluated using a modified TRAP-eze (Intergen) detection protocol. At the time of each endometrial biopsy, serum estrogen and progesterone were measured. Proliferative phase endometrium showed high telomerase activity. At the onset of the luteal phase telomerase activity was high, but it decreased during the early luteal phase, disappeared by the midluteal phase (6 d after LH surge detected), and then rose to moderate levels in the late luteal phase beginning on luteal d 10. Serum progesterone levels were inversely related to telomerase activity.

In conclusion, endometrial telomerase activity is dynamic: high during the proliferative phase but inhibited during the midsecretory phase of the menstrual cycle. The timing of expression coincides with the rise and fall of progesterone levels and the time period of maximal uterine receptivity for embryo implantation. This supports a relationship between sex steroid levels and telomerase regulation.
Ca2+/Nicotinamide Adenine Dinucleotide Phosphate-Dependent H2O2 Generation Is Inhibited by Iodide in Human Thyroids
Authors: Cardoso Luciene C. , Martins Denise C. L. , Figueiredo Marcia D. L. , Rosenthal Doris , Vaisman Mario , Violante Alice H. D. , Carvalho Denise P.
Published: January 2014

A calcium and NAD(P)H-dependent H2O2-generating activity has been studied in paranodular thyroid tissues from four patients with cold thyroid nodules and from nine diffuse toxic goiters. H2O2 generation was detected both in the particulate (P 3,000 g) and in the microsomal (P 100,000 g) fractions of paranodular tissue surrounding cold thyroid nodules (PN), with the same biochemical properties described for NADPH oxidase found in porcine and human thyroids. In PN tissues, the particulate NADPH oxidase activity (224 ± 38 nmol H2O2·h−1·mg−1 protein) was similar to that described for the porcine thyroid enzyme. However, no NADPH oxidase activity was detectable in the particulate fractions from eight diffuse toxic goiter patients treated with iodine before surgery; all but one also received propylthiouracil or methimazole in the preoperative period. Thyroid cytochrome c reductase (diffuse toxic goiters = 438 ± 104 nmol NADP+·h−1·mg−1 protein; PN = 78 ± 10 nmol NADP+·h−1·mg−1 protein) and thyroperoxidase (diffuse toxic goiters = 621 ± 179 U·g−1 protein; PN = 232 ± 121 U·g−1 protein) activities were unaffected by iodide. Thus, the human NADPH oxidase seems to be inhibited by iodinated compounds in vivo and probably is an enzyme involved in the Wolff-Chaikoff effect. Our findings reinforce the hypothesis that thyroid NADPH oxidase is responsible for the production of H2O2 necessary for thyroid hormone biosynthesis.
The Effects of Soy-Derived Phytoestrogens on Serum Lipids and Lipoproteins in Moderately Hypercholesterolemic Postmenopausal Women
Authors: Dewell Antonella , Hollenbeck Clarie B. , Bruce Bonnie
Published: January 2014

Postmenopausal women are at an increased risk of developing coronary artery disease (CAD). This increase is due primarily to elevated cholesterol concentrations accompanying the loss of endogenous estrogen secretion. Recently, the consumption of soy foods has been shown to reduce serum cholesterol concentrations. Phytoestrogens (PE) have been proposed as the responsible agents of the hypocholesterolemic effect of soy foods. However, few studies have investigated the effect of PE supplementation on serum lipoproteins. The purpose of the present study is to investigate the effects of PE supplementation (150 mg) on serum lipids and lipoproteins in moderately hypercholesterolemic, elderly, postmenopausal women. Thirty-six subjects were randomized into two groups and received either a 150-mg PE supplement/d (n = 20) or a placebo (n = 16). Serum samples obtained at baseline and 2 months were analyzed for total triacylglycerol, total cholesterol, and high density lipoprotein cholesterol using standard Lipid Research Clinic procedures. In addition, total triacylglycerol and cholesterol were measured after 6 months of treatment. The t test and ANOVA were employed to compare the two groups. The results (mean ± sem) indicated no significant differences in total triacylglycerol (1.3 ± 0.2 vs. 1.2 ± 0.2 mmol/liter), total cholesterol (6.4 ± 0.4 vs. 6.5 ± 0.2 mmol/liter), or high density lipoprotein cholesterol (1.0 ± 0.1 vs. 1.0 ± 0.1 mmol/liter) between the placebo and the PE groups, respectively, after 2 months of treatment. Moreover, total triacylglycerol and cholesterol remained unchanged after 6 months. Our findings suggest that PE supplementation with 150 mg/d over a 6-month period does not significantly alter serum lipoproteins in postmenopausal women and, therefore, may not effectively reduce the risk of CAD in this population.
Cardiac Involvement in Thyroid Hormone Resistance
Authors: Kahaly George J. , Matthews Clare H. , Mohr-Kahaly Susanne , Richards Chris A. , Chatterjee V. Krishna K.
Published: January 2014

To analyze the cardiovascular alterations thought to occur in resistance to thyroid hormone (RTH), cardiac involvement in 54 patients with RTH was investigated with the help of two-dimensional and Doppler echocardiography. Data from 41 of 54 adult subjects with RTH were also compared with those of 24 and 20 cases with hyperthyroidism (H) and hypothyroidism (h), respectively, as well as 22 healthy euthyroid controls (C). With respect to the type of mutations, no correlation was found between cardiovascular features and genotype. Compared with affected adults, children with RTH showed markedly higher serum free T3 (FT3), free T4 (FT4), and baseline TSH concentrations. Compared with healthy children of comparable age, RTH children had significantly higher heart rate and lower left ventricular (LV) ejection fraction (P = 0.006). Also, higher heart rate and FT4 as well as shorter diastolic relaxation of the myocardium (all P = 0.001) between RTH subjects with and without thyrotoxic cardiovascular features were found. Cardiac symptoms (palpitations, 32% vs. 71%) and signs (sinus tachycardia, 26% vs. 79%; atrial fibrillation, 6% vs. 17%) were significantly less frequent in RTH vs. H (all P = 0.001). Compared with C and h, heart rate, cardiac output, stroke volume, and systolic aortic flow velocity were strongly increased in RTH (all P = 0.0001) and H, although ejection (P = 0.0012) and shortening (P = 0.0001) fractions of the LV were markedly lower in RTH vs. H. Diastolic parameters, such as isovolumic relaxation (P = 0.0001) and deceleration time (P = 0.013), were shorter in RTH vs. h and C. In RTH, positive correlations between FT3 and heart rate, and between FT4 and LV ejection fraction were observed, whereas negative correlations between both FT3 and FT4 and isovolumic relaxation were noted. In conclusion, these findings indicate a modulated hyperthyroid effect on cardiac systolic and diastolic function of the myocardium in RTH, whereas other parameters, such as ejection and shortening fractions of the LV, systolic diameter, and LV wall thickness, were comparable to C. Differences in term of cardiovascular changes were smaller between the RTH and C groups than the RTH and the H or h groups. Thus, an incomplete cardiac response to thyroid hormone is present in RTH.
Spironolactone Abolishes the Relationship between Aldosterone and Plasminogen Activator Inhibitor-1 in Humans
Authors: Sawathiparnich Pairunyar , Kumar Sandeep , Vaughan Douglas E. , Brown Nancy J.
Published: January 2014

Recent studies have defined a link between the renin-angiotensin-aldosterone system and fibrinolysis. The present study tests the hypothesis that endogenous aldosterone regulates plasminogen activator inhibitor-1 (PAI-1) production in humans. Hemodynamic parameters, PAI-1 and tissue-type plasminogen activator (t-PA) antigen, potassium, PRA, angiotensin II, and aldosterone were measured in nine male hypertensive subjects after a 3-wk washout, after 2 wk of hydrochlorothiazide (HCTZ; 25 mg plus 20 mmol KCl/d), and after 2 wk of spironolactone (100 mg/d plus KCl placebo). Spironolactone (P = 0.04), but not HCTZ (P = 0.57 vs. baseline; P = 0.1 vs. spironolactone), significantly lowered systolic blood pressure. Angiotensin II increased from baseline during both HCTZ (P = 0.02) and spironolactone (P = 0.02 vs. baseline; P = 0.19 vs. HCTZ) treatments. Although both HCTZ (P = 0.004) and spironolactone (P < 0.001 vs. baseline) increased aldosterone, the effect was greater with spironolactone (P < 0.001 vs. HCTZ). HCTZ increased PAI-1 antigen (P = 0.02), but did not alter t-PA antigen. In contrast, there was no effect of spironolactone on PAI-1 antigen (P = 0.28), whereas t-PA antigen was increased (P = 0.01). There was a significant correlation between PAI-1 antigen and serum aldosterone during both baseline and HCTZ study days (r2 = 0.57; P = 0.0003); however, treatment with spironolactone abolished this correlation (r2 = 0.13; P = 0.33). This study provides evidence that endogenous aldosterone influences PAI-1 production in humans.
Effects of Recombinant Human Growth Hormone on Hepatic Lipid and Carbohydrate Metabolism in HIV-Infected Patients with Fat Accumulation
Authors: Schwarz Jean-Marc , Mulligan Kathleen , Lee Jeongae , Lo Joan C. , Wen Michael , Noor Mustafa A. , Grunfeld Carl , Schambelan Morris
Published: January 2014

We recently reported that treatment with a pharmacologic dose of recombinant human growth hormone (GH) resulted in a significant loss of body fat and gain in lean tissue in HIV-infected patients with syndromes of fat accumulation. However, insulin-mediated glucose disposal decreased transiently after one month of GH therapy. The present paper focuses on the changes of hepatic carbohydrate and fat metabolism associated with GH treatment in the same subjects. We assessed hepatic insulin sensitivity under both fasting and hyperinsulinemic-euglycemic clamp conditions prior to and after one and six months of GH treatment (3 mg/day) in five patients using stable isotope tracer techniques. Indirect calorimetry, and measurements of lipid concentrations. Fasting endogenous glucose production (EGP) increased significantly at one month (12.0 ± 0.7 to 14.9 ± 0.9 μmol/kg/min, P < 0.03), and the increase was sustained at six months of GH treatment (14.0 ± 1.1μ mol/kg/min, NS). This increase in EGP was driven in part by increased glucogenesis (GNG) (3.5 ± 0.9 to 5.2 ± 0.9 and 5.8 ±1.2 μmol/kg/min, n = 4, P < 0.01 and P < 0.01 at one and six months, respectively); small changes in hepatic glycogenolysis also contributed. Sustained increases in lipolysis and progressive decreases in hepatic fractional de novo lipogenesis (DNL) and triglyceride concentrations occurred with GH treatment. These changes were accompanied by an improved lipid profile with a significant increase in HDL cholesterol and significant decreases in total and LDL cholesterol and triglyceride levels, the latter consistent with the decrease in hepatic DNL. During a hyperinsulinemic-euglycemic glucose clamp, EGP and GNG were markedly suppressed compared to the corresponding time points under fasting conditions, albeit less so when measured after one month of GH treatment. Thus, in HIV-infected patients with abnormal fat distribution, pharmacologic doses of GH improved the overall lipid profile, but worsened glucose homeostasis under both fasting and hyperinsulinemic conditions. The combined implications of these positive and negative metabolic effects for cardiovascular disease risk remain unknown.
Measurements of Interstitial Muscle Glycerol in Normal and Insulin-Resistant Subjects
Authors: Sjöstrand Mikaela , Gudbjörnsdottir Soffia , Holmäng Agneta , Strindberg Lena , Ekberg Karin , Lönnroth Peter
Published: January 2014

The aim of this project was to study the regulation of interstitial glycerol levels in muscle in normal subjects, and to estimate interstitial muscle glycerol in obese subjects and patients with type 2 diabetes. In healthy lean subjects, microdialysis of forearm sc and muscle tissue were combined with arterial and deep venous catheterization, as well as blood flow registrations during oral glucose ingestion. In two other separate studies, obese (n = 9) vs. lean (n = 10) subjects and type 2 diabetes patients (n = 8) vs. weight-matched control subjects (n = 8) were investigated by means of muscle microdialysis during a euglycemic hyperinsulinemic clamp. Oral glucose ingestion suppressed the interstitial sc glycerol concentration by approximately 40% (P < 0.05), whereas no significant reduction of muscle interstitial glycerol was found. In contrast to the significant muscle interstitial-arterial (I-A) glycerol difference, the venous-arterial difference was small and varying throughout the oral glucose tolerance test. At steady-state hyperinsulinemia, obese subjects’ interstitial muscle glycerol and I-A glycerol difference were both significantly higher than lean controls, whereas type 2 diabetes patient had interstitial muscle glycerol concentrations and I-A glycerol differences similar to those found in weight-matched controls. A significant and marked I-A glycerol difference exists in the absence of a significant venous-arterial difference, indicating that muscle glycerol cannot be taken as a marker of intramyocellular lipolysis because local turnover of muscle glycerol might be significant. The present data also suggest that, in contrast to sc tissue, muscle tissue lacks a clear antilipolytic effect of insulin. Moreover, the muscle interstitial glycerol concentration is elevated in obese patients but does not precipitate insulin resistance and type 2 diabetes.
Growth Differentiation Factor-9 Inhibits 3′5′-Adenosine Monophosphate-Stimulated Steroidogenesis in Human Granulosa and Theca Cells
Authors: Yamamoto Noriko , Christenson Lane K. , McAllister Jan M. , Strauss Jerome F.
Published: January 2014

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor superfamily, modulates the development and function of granulosa and theca cells. Targeted deletion of GDF-9 in the mouse revealed that GDF-9 was essential for the establishment of the thecal cell layer during early folliculogenesis. During later stages of follicular development, the roles of GDF-9 are less well understood, but it has been postulated that oocyte-derived GDF-9 may prevent premature luteinization of follicular cells, based on its ability to modulate steroidogenesis by rodent ovarian cells. In the rodent, GDF-9 is expressed solely by the oocyte from the early primary follicular stage through ovulation. Recent studies in the rhesus monkey demonstrated that granulosa cells express GDF-9, suggesting a broader role for this protein in ovarian function in primates. We examined the effect of recombinant GDF-9 on proliferating human granulosa and thecal cell steroidogenesis and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage, and P450 aromatase. We also examined granulosa cell GDF-9 expression by quantitative RT-PCR and by Western analysis. GDF-9 inhibited 8-Br-cAMP-stimulated granulosa progesterone synthesis by approximately 40%, but did not affect basal progesterone production. Concordant with reduced steroid production, 8-Br-cAMP-stimulated StAR protein expression was reduced approximately 40% in granulosa cells, as were expression of StAR mRNA and StAR promoter activity. Additionally, GDF-9 inhibited 8-Br-cAMP-stimulated expression of P450 side-chain cleavage and P450 aromatase. Human granulosa cells expressed GDF-9, as determined by RT-PCR and Western analysis. Treatment of human thecal cells with GDF-9 blocked forskolin-stimulated progesterone, 17α-hydroxyprogesterone, and dehydroepiandrosterone synthesis. Thecal cells exhibited greater sensitivity to GDF-9, suggesting that this cell may be a primary target of GDF-9. Moreover, GDF-9 increased thecal cell numbers during culture, but had no effect on granulosa cell growth. Our findings implicate GDF-9 in the modulation of follicular steroidogenesis, especially theca cell function. Because GDF-9 mRNA and protein are detectable in granulosa-lutein cells after the LH surge, the concept of GDF-9 as a solely oocyte-derived luteinization inhibitor needs to be reevaluated.
Effect of Pioglitazone on Abdominal Fat Distribution and Insulin Sensitivity in Type 2 Diabetic Patients
Authors: Miyazaki Yoshinori , Mahankali Archana , Matsuda Masafumi , Mahankali Srikanth , Hardies Jean , Cusi Kenneth , Mandarino Lawrence J. , DeFronzo Ralph A.
Published: January 2014

We examined the effect of pioglitazone on abdominal fat distribution to elucidate the mechanisms via which pioglitazone improves insulin resistance in patients with type 2 diabetes mellitus. Thirteen type 2 diabetic patients (nine men and four women; age, 52 ± 3 yr; body mass index, 29.0 ± 1.1 kg/m2), who were being treated with a stable dose of sulfonylurea (n = 7) or with diet alone (n = 6), received pioglitazone (45 mg/d) for 16 wk. Before and after pioglitazone treatment, subjects underwent a 75-g oral glucose tolerance test (OGTT) and two-step euglycemic insulin clamp (insulin infusion rates, 40 and 160 mU/m2·min) with [3H]glucose. Abdominal fat distribution was evaluated using magnetic resonance imaging at L4–5. After 16 wk of pioglitazone treatment, fasting plasma glucose (179 ± 10 to 140 ± 10 mg/dl; P < 0.01), mean plasma glucose during OGTT (295 ± 13 to 233 ± 14 mg/dl; P < 0.01), and hemoglobin A1c (8.6 ± 0.4% to 7.2 ± 0.5%; P < 0.01) decreased without a change in fasting or post-OGTT insulin levels. Fasting plasma FFA (674 ± 38 to 569 ± 31 μEq/liter; P < 0.05) and mean plasma FFA (539 ± 20 to 396 ± 29 μEq/liter; P < 0.01) during OGTT decreased after pioglitazone. In the postabsorptive state, hepatic insulin resistance [basal endogenous glucose production (EGP) × basal plasma insulin concentration] decreased from 41 ± 7 to 25 ± 3 mg/kg fat-free mass (FFM)·min × μU/ml; P < 0.05) and suppression of EGP during the first insulin clamp step (1.1 ± 0.1 to 0.6 ± 0.2 mg/kg FFM·min; P < 0.05) improved after pioglitazone treatment. The total body glucose MCR during the first and second insulin clamp steps increased after pioglitazone treatment [first MCR, 3.5 ± 0.5 to 4.4 ± 0.4 ml/kg FFM·min (P < 0.05); second MCR, 8.7 ± 1.0 to 11.3 ± 1.1 ml/kg FFM·min (P < 0.01)]. The improvement in hepatic and peripheral tissue insulin sensitivity occurred despite increases in body weight (82 ± 4 to 85 ± 4 kg; P < 0.05) and fat mass (27 ± 2 to 30 ± 3 kg; P < 0.05). After pioglitazone treatment, sc fat area at L4–5 (301 ± 44 to 342 ± 44 cm2; P < 0.01) increased, whereas visceral fat area at L4–5 (144 ± 13 to 131 ± 16 cm2; P < 0.05) and the ratio of visceral to sc fat (0.59 ± 0.08 to 0.44 ± 0.06; P < 0.01) decreased. In the postabsorptive state hepatic insulin resistance (basal EGP × basal immunoreactive insulin) correlated positively with visceral fat area (r = 0.55; P < 0.01). The glucose MCRs during the first (r = −0.45; P < 0.05) and second (r = −0.44; P < 0.05) insulin clamp steps were negatively correlated with the visceral fat area. These results demonstrate that a shift of fat distribution from visceral to sc adipose depots after pioglitazone treatment is associated with improvements in hepatic and peripheral tissue sensitivity to insulin.
Identification of a Novel Sonic Hedgehog Response Element in the Chicken Ovalbumin Upstream Promoter-Transcription Factor II Promoter
Authors: Krishnan Venkatesh , Elberg Gerard , Tsai Ming-Jer , Tsai Sophia Y.
Published: January 2014

Sonic hedgehog (Shh) is a secreted morphogen that regulates dorso-ventral patterning within the neural tube during embryonic development. It is well established that Shh can induce motor-neuron differentiation that coincides with the appearance of specific motor-neuron markers including chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) and Isl1. However, the mechanism of Shh-induced signaling pathway in vertebrates is not clearly defined. In this report we have identified COUP-TFII as a target gene for Shh. In addition we have used a 1.6-kb region of the COUP-TFII promoter to identify a target element that mediates the Shh-induced activity. Extensive deletions introduced within this region have further enabled us to identify a novel sonic hedgehog response element (ShhRE) in the COUP-TFII promoter. Point mutations introduced within the ShhRE reveal some key nucleotides that are essential for protein(s)-binding activity. Finally, the ShhRE is capable of functioning as a true enhancer element and can mediate Shh-induced transactivation of reporter gene via a heterologous promoter.
Insulin-Like Growth Factor I Regulates Gonadotropin Responsiveness in the Murine Ovary
Authors: Zhou Jian , Kumar T. Rajendra , Matzuk Martin M. , Bondy Carolyn
Published: January 2014

The present study shows that insulin-like growth factor I (IGF-I) and FSH receptor (FSHR) mRNAs are selectively coexpressed in a subset of healthy-appearing follicles in murine ovaries, irrespective of cycle stage. Aromatase gene expression, a prime marker for FSH effect, is found only in IGF-I/FSHR- positive follicles, showing that these are healthy, gonadotropin-responsive follicles. Given the striking coexpression of FSHR and IGF-I, we hypothesized that FSH was responsible for follicular IGF-I expression. We found, however, that granulosa cell IGF-I mRNA levels are not reduced in hypophysectomized (±PMSG) or FSH knockout mice, indicating that FSH does not have a major role in regulation of granulosa cell IGF-I gene expression. To test the alternative hypothesis that IGF-I regulates FSHR gene expression, we studied ovaries from IGF-I knockout mice. FSHR mRNA was significantly reduced in ovaries from homozygous IGF-I knockout compared with wild type mice and was restored to control values by exogenous IGF-I treatment. The functional significance of the reduced FSHR gene expression in IGF-I knockout ovaries is suggested by reduced aromatase expression and by the failure of their follicles to develop normally beyond the early antral stage. In fact, IGF-I knockout and FSH knockout ovaries appear very similar in terms of arrested follicular development. In summary, we have shown that IGF-I and FSHR are selectively coexpressed in healthy, growing murine follicles and that FSH does not affect IGF-I expression but that IGF-I augments granulosa cell FSHR expression. These data suggest that ovarian IGF-I expression serves to enhance granulosa cell FSH responsiveness by augmenting FSHR expression.
A Homozygous Microdeletion in Helix 7 of the Luteinizing Hormone Receptor Associated with Familial Testicular and Ovarian Resistance Is Due to Both Decreased Cell Surface Expression and Impaired Effector Activation by the Cell Surface Receptor
Authors: Latronico Ana C. , Chai Yaohui , Arnhold Ivo J.P. , Liu Xuebo , Mendonca Berenice B. , Segaloff Deborah L.
Published: January 2014

In this report, the genomic DNA was examined from two siblings with gonadal LH resistance. A 46,XY pseudohermaphrodite presented with female external genitalia and his 46,XX sister exhibited menstrual irregularities (oligoamenorrhea) and infertility. Exons 1–11 of the LH receptor (LHR) gene were amplified by the PCR using different sets of intronic primers and were directly sequenced. Sequencing revealed that both individuals carried a deletion of nucleotides 1822–1827, resulting in the deletion of Leu-608 and Val-609 within the seventh transmembrane helix. This mutation was introduced into a recombinant human (h) LHR cDNA. Transfections of 293 cells with hLHR(wt) vs. hLHR(ΔL608,V609) revealed that very little of the mutant receptor was expressed at the cell surface. This was due to both a decrease in the total amount of receptor expressed as well as to an increased intracellular retention of the mutant receptor. In spite of the decreased cell surface expression of the mutant, sufficient amounts were present to allow for assessment of its functions. Equilibrium binding assays showed that the cell surface hLHR(ΔL608,V609) binds hCG with an affinity comparable to that of the wild-type receptor. However, the cells expressing the hLHR(ΔL608,V609) exhibit only a 1.5- to 2.4-fold stimulation of cAMP production in response to hCG. In contrast, cells expressing comparably low levels of hLHR(wt) responded to hCG with 11- to 30-fold increases of cAMP levels. Therefore, the testicular and ovarian unresponsiveness to LH in these patients appears to be due to a mutation of the hLHR gene in which Leu-608 and Val-609 are deleted. As a consequence, the majority of the mutant receptor is retained intracellularly. The small percentage of mutant receptor that is expressed at the cell surface binds hormone normally but is unable to activate Gs.
Transcription of the Leydig Insulin-Like Gene Is Mediated by Steroidogenic Factor-1
Authors: Zimmermann Stephan , Schwärzler Anja , Buth Sabine , Engel Wolfgang , Adham Ibrahim M.
Published: January 2014

The Leydig insulin-like gene (Ley I-L), a member of the insulin-related gene family, is specifically expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. To determine the functional region of the mouse Ley I-L promoter and factors controlling the Ley I-L gene expression, we used 2.1 kb of the 5′-flanking region of the mouse Ley I-L gene to generate chimeric constructs with the chloramphenicol acetyltransferase gene (CAT). Transient transfections of MA10 Leydig cells, LTK− fibroblasts, and F9 embryonic cells by a series of 5′-deleted mouse Ley I-L promoter-CAT constructs revealed that the sequence between nucleotides −157 to +4 directs the transcription of the reporter gene in MA10 but not in LTK− and F9 cells, indicating that the determinants of Leydig cell-specific expression reside within this region. Deoxyribonuclease I (DNase I) footprint analysis revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase I-protected regions are homologous to the consensus binding site of the steroidogenic factor-1 (SF-1). Competition and antibody studies showed that the three SF-1-binding sites in the Ley I-L promoter have similar binding affinities for SF-1. Furthermore, transient transfections of MA10 cells with mutant reporter constructs, in which SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1- binding sites are required for SF-1-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing expression vector together with a Ley I-L promoter-CAT construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicated that SF-1 can transactivate the Ley I-L promoter. These data demonstrate an essential role of SF-1 in transcriptional activation of the Ley I-L promoter.
Caveolin-1 Interacts with the Insulin Receptor and Can Differentially Modulate Insulin Signaling in Transfected Cos-7 Cells and Rat Adipose Cells
Authors: Nystrom Fredrik H. , Chen Hui , Cong Li-Na , Li Yunhua , Quon Michael J.
Published: January 2014

Caveolae may function as microdomains for signaling that help to determine specific biological actions mediated by the insulin receptor (IR). Caveolin-1, a major component of caveolae, contains a scaffolding domain (SD) that binds to a caveolin-1 binding motif in the kinase domain of the IR in vitro. To investigate the potential role of caveolin-1 in insulin signaling we overexpressed wild-type (Cav-WT) or mutant (Cav-Mut; F92A/V94A in SD) caveolin-1 in either Cos-7 cells cotransfected with IR or rat adipose cells (low and high levels of endogenous caveolin-1, respectively). Cav-WT coimmunoprecipitated with the IR to a much greater extent than Cav-Mut, suggesting that the SD is important for interactions between caveolin-1 and the IR in intact cells. We also constructed several IR mutants with a disrupted caveolin-1 binding motif and found that these mutants were poorly expressed and did not undergo autophosphorylation. Interestingly, overexpression of Cav-WT in Cos-7 cells significantly enhanced insulin-stimulated phosphorylation of Elk-1 (a mitogen-activated protein kinase-dependent pathway) while overexpression of Cav-Mut was without effect. In contrast, in adipose cells, overexpression of either Cav-WT or Cav-Mut did not affect insulin-stimulated phosphorylation of a cotransfected ERK2 (but did significantly inhibit basal phosphorylation of ERK2). Furthermore, we also observed a small inhibition of insulin-stimulated translocation of GLUT4 when either Cav-WT or Cav-Mut was overexpressed in adipose cells. Thus, interaction of caveolin-1 with IRs may differentially modulate insulin signaling to enhance insulin action in Cos-7 cells but inhibit insulin’s effects in adipose cells.
Growth Arrest-Specific Gene 6 (Gas6)/Adhesion Related Kinase (Ark) Signaling Promotes Gonadotropin-Releasing Hormone Neuronal Survival via Extracellular Signal-Regulated Kinase (ERK) and Akt
Authors: Allen Melissa P. , Zeng Chan , Schneider Kristina , Xiong Xiaoyan , Meintzer Mary Kay , Bellosta Paola , Basilico Claudio , Varnum Brian , Heidenreich Kim A. , Wierman Margaret E.
Published: January 2014

We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1–7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Axl) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1–7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Axl) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration.