bluuebunny/crossref_metadata_2025 huggingface dataset:
Decrease in Carotid Intima-Media Thickness after 1-Year Therapy with Etidronate for Osteopenia Associated with Type 2 Diabetes1
Authors: Koshiyama Hiroyuki , Nakamura Yoshio , Tanaka Satsuki , Minamikawa Jun
Published: January 2014

It has been suggested that bisphosphonates may have some antiatherogenic actions in experimental animals or in vitro, but their effects on the atherogenic process in humans has not been reported. In the present study the effect of etidronate treatment on carotid arterial intima-media thickness was prospectively examined in 57 subjects with type 2 diabetes associated with osteopenia. After 1 yr of therapy with cyclical etidronate (200 mg/day for 2 weeks every 3 months), intima-media thickness showed a decrease (mean ± se, −0.038 ± 0.011 mm), which was significantly different from a change in 57 control subjects (0.023 ± 0.015 mm; P < 0.005). Cardiovascular parameters were not changed after etidronate treatment. These findings suggest that etidronate in clinical dosage may have an antiatherogenic action, at least in type 2 diabetes, although its mechanisms remain to be elucidated.
Sex-Determining Gene(s) on Distal 9p: Clinical and Molecular Studies in Six Cases*
Authors: Muroya Koji , Okuyama Torayuki , Goishi Keiji , Ogiso Yoshifumi , Fukuda Shin , Kameyama Junji , Sato Hirokazu , Suzuki Yoshimi , Terasaki Hiroshi , Gomyo Hiroki , Wakui Keiko , Fukushima Yoshimitsu , Ogata Tsutomu
Published: January 2014

We report on clinical and molecular findings in five karyotypic males (cases 1–5) and one karyotypic female (case 6) with distal 9p monosomy. Cases 1–3 and 6 had female external genitalia, case 4 showed ambiguous external genitalia, and case 5 exhibited male external genitalia with left cryptorchidism and right intrascrotal testis. Gonadal explorations at gonadectomy in cases 3 and 4 revealed that case 3 had left streak gonad and right agonadism, and case 4 had bilateral hypoplastic testes. Endocrine studies in cases 1–4 and 6 showed that cases 1, 3, and 6 had definite primary hypogonadism, with basal FSH levels of 54, 39, and 41 IU/L, respectively, whereas case 2 with severe malnutrition was unremarkable for the baseline values, and case 4 had fairly good testicular function. Fluorescence in situ hybridization and microsatellite analyses demonstrated that all cases had hemizygosity of the 9p sex-determining region distal to D9S1779, with loss of the candidate sex-determining genes DMRT1 and DMRT2 from the abnormal chromosome 9. Sequence analysis in cases 1–4 and 6 showed that they had normal sequences of each exon of DMRT1 and the DM domain of DMRT2 on the normal chromosome 9, and that cases 1–4 had normal SRY sequence.

The results provide further support for the presence of a sex-determining gene(s) on distal 9p and favor the possibility of DMRT1 and/or DMRT2 being the sex-determining gene(s). Furthermore, as hemizygosity of the 9p sex-determining region was associated with a wide spectrum of gonadogenesis from agonadism to testis formation in karyotypic males and with primary hypogonadism regardless of karyotypic sex, it is inferred that haploinsufficiency of the 9p sex-determining gene(s) primarily hinders the formation of indifferent gonad, leading to various degrees of defective testis formation in karyotypic males and impaired ovary formation in karyotypic females.
Stimulatory Effect of Adrenocorticotropin on Cortisol, Aldosterone, and Dehydroepiandrosterone Secretion in Normal Humans: Dose-Response Study*
Authors: Arvat Emanuela , Di Vito Lidia , Lanfranco Fabio , Maccario Mauro , Baffoni Claudia , Rossetto Ruth , Aimaretti Gianluca , Camanni Franco , Ghigo Ezio
Published: January 2014

The short ACTH test is widely used in clinical practice for the diagnosis of adrenal insufficiency. It is classically performed administering 250.0 μg ACTH(1–24) although 1.0 μg ACTH dose has been reported having maximal stimulatory effect on cortisol levels in normal subjects. We aimed to define the maximal and the minimal stimulatory ACTH dose on cortisol, aldosterone, and dehydroepiandrosterone (DHEA) in humans. To this goal, in 12 normal volunteers (6 males and 6 females; age, 22–34 yr; body mass index 20–25 kg/m2; body surface 1.6–1.9 m2), we studied the dose-response effect of eight ACTH doses (0.01, 0.03, 0.06, 0.125, 0.5, 1.0, 25.0, and 250.0 μg) on cortisol, aldosterone, and DHEA levels. Each ACTH dose administered at 0 min was followed by a second ACTH dose of 250.0 μg at +60 min. The cortisol Δ areas under response curve (ΔAUCs) after all ACTH doses, apart from 0.01 μg, were significantly higher (P < 0.02) than that after placebo, showing a clear dose-response relationship (P< 0.001). The doses of 0.03 and 1.0 μg ACTH were the minimal and maximal effective doses, respectively. The cortisol response to 250.0μ g ACTH was not modified by pretreatment with 0.01, 0.03, and 0.06μ g ACTH doses, whereas it was progressively reduced by increasing the dose of ACTH pretreatment (P < 0.001). The aldosteroneΔ AUCs to all but 0.01 μg ACTH doses were significantly higher (P < 0.02) than that after placebo, showing a clear dose-response relationship (P < 0.001). The dose of 0.03 μg was the minimal effective stimulating dose, whereas 25.0 μg showed the same aldosterone-releasing effect of 250.0 μg. The aldosterone response to 250.0 μg ACTH, preceeded by placebo, was not modified by pretreatment with 0.01 and 0.03 μg ACTH doses, whereas it was reduced by increasing the dose of ACTH pretreatment (P < 0.05–0.02). The DHEA ΔAUCs to all ACTH doses were significantly higher (P < 0.01) than that after placebo, showing a clear dose-response relationship (P< 0.001). The doses of 0.01 and 1.0 μg ACTH were the minimal and maximal effective dose, respectively. The DHEA response to 250.0 μg ACTH was not modified by pretreatment with 0.01, 0.03, 0.06, and 0.125μ g ACTH doses, whereas it was progressively reduced by pretreatment with 0.5, 1.0, and 25.0 μg ACTH doses (P < 0.01). In conclusion, these results show that an extremely low ACTH dose is needed to stimulate adrenal steroids and, among them, DHEA seems the most sensitive to corticotropin stimulation.
Ovarian Response to Follicle-Stimulating Hormone (FSH) Stimulation Depends on the FSH Receptor Genotype*
Authors: Mayorga Maritza Perez , Gromoll Jörg , Behre Hermann M. , Gassner Claudia , Nieschlag Eberhard , Simoni Manuela
Published: January 2014

Because the ovarian response to FSH stimulation in assisted reproduction is variable, ranging from hyporesponse to hyperresponse, with the possible complication of ovarian hyperstimulation, it would be of great benefit to predict the response of the patients to FSH. To date, no clear-cut predictors of ovarian responsiveness to FSH have been identified. In this study, we investigated the role of two distinct FSH receptor (FSHR) variants, Thr307/Asn680 and Ala307/Ser680, in the response to FSH in women undergoing controlled ovarian stimulation.

The FSHR polymorphism at position 680 was analyzed by restriction-fragment-length polymorphism in 161 ovulatory women below the age of 40 yr. With reference to the couple, infertility has been diagnosed as being attributable to male causes (76%), tubal factor (11%), or both (13%). The distribution was 29% for the Asn/Asn, 45% for the Asn/Ser, and 26% for the Ser/Ser FSHR variant. Peak estradiol levels, number of preovulatory follicles, and number of retrieved oocytes were similar in the 3 groups. However, basal FSH levels were significantly different among the 3 groups (6.4 ± 0.4 IU/L, 7.9 ± 0.3 IU/L, and 8.3 ± 0.6 IU/L for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.01). The number of FSH ampoules required for successful stimulation was significantly different among the 3 groups (31.8 ± 2.4, 40.7 ± 2.3, and 46.8 ± 5.0 for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.05). According to multiple linear regression analysis, the number of ampoules needed could be predicted from a linear combination of both the type of polymorphism and basal FSH levels (P < 0.001).

These clinical findings demonstrate that the ovarian response to FSH stimulation depends on the FSHR genotype.
Stomach Is a Major Source of Circulating Ghrelin, and Feeding State Determines Plasma Ghrelin-Like Immunoreactivity Levels in Humans
Authors: Ariyasu Hiroyuki , Takaya Kazuhiko , Tagami Tetsuya , Ogawa Yoshihiro , Hosoda Kiminori , Akamizu Takashi , Suda Michio , Koh Toshikiyo , Natsui Koshi , Toyooka Shigetake , Shirakami Gotaro , Usui Takeshi , Shimatsu Akira , Doi Kentaro , Hosoda Hiroshi , Kojima Masayasu , Kangawa Kenji , Nakao Kazuwa
Published: January 2014

Ghrelin, an endogenous ligand for the GH secretagogue receptor, was isolated from rat stomach and is involved in a novel system for regulating GH release. Although previous studies in rodents suggest that ghrelin is also involved in energy homeostasis and that ghrelin secretion is influenced by feeding, little is known about plasma ghrelin in humans. To address this issue, we studied plasma ghrelin-like immunoreactivity levels and elucidated the source of circulating ghrelin and the effects of feeding state on plasma ghrelin-like immunoreactivity levels in humans. The plasma ghrelin-like immunoreactivity concentration in normal humans measured by a specific RIA was 166.0 ± 10.1 fmol/ml. Northern blot analysis of various human tissues identified ghrelin mRNA found most abundantly in the stomach and plasma ghrelin-like immunoreactivity levels in totally gastrectomized patients were reduced to 35% of those in normal controls. Plasma ghrelin-like immunoreactivity levels were increased by 31% after 12-h fasting and reduced by 22% immediately after habitual feeding. In patients with anorexia nervosa, plasma ghrelin-like immunoreactivity levels were markedly elevated compared with those in normal controls (401.2 ± 58.4 vs. 192.8 ± 19.4 fmol/ml) and were negatively correlated with body mass indexes. We conclude that the stomach is a major source of circulating ghrelin and that plasma ghrelin-like immunoreactivity levels reflect acute and chronic feeding states in humans.
Increased Final Height in Precocious Puberty after Long-Term Treatment with LHRH Agonists: The National Institutes of Health Experience
Authors: Klein Karen Oerter , Barnes Kevin M. , Jones Janet V. , Feuillan Penelope P. , Cutler Jr. Gordon B.
Published: January 2014

We report 98 children who have reached final adult height in a long-term trial of LHRH agonist treatment. These children were 5.3± 2.1 yr old at the start of treatment and were treated with either deslorelin (4 μg/kg·d sc) or histrelin (4–10 μg/kg·d) for an average of 6.1 ± 2.5 yr. Final height averaged 159.8 ± 7.6 cm in the 80 girls, which was significantly greater than pretreatment predicted height (149.3 ± 9.6 cm) but still significantly less than midparental height (MPH) (163.7 ± 5.6). Final height averaged 171.1 ± 8.7 cm in the 18 boys, which was significantly greater than pretreatment predicted height (156.1 ± 14.2 cm) but still significantly less than MPH (178.3 ± 5.2 cm). However, the average adult height of the 54 children who had less than a 2-yr delay in the onset of treatment was not significantly different from their MPH, and 21 children exceeded MPH.

Final height sd score correlated positively with duration of treatment (P < 0.01), midparental height (P < 0.001), predicted height at the start of treatment (P < 0.001), and growth velocity during the last year of treatment (P < 0.001) and correlated inversely with delay in the onset of treatment (P < 0.001), age at the start of treatment (P < 0.001), bone age at the start of treatment (P < 0.001), bone age at the end of treatment (P < 0.001), breast stage at the start of treatment (P = 0.02), and bone age minus chronological age at the start of treatment (P = 0.001).

We conclude that LHRH agonist treatment improves the final height for children with rapidly progressing precocious puberty treated before the age of 8 yr for girls or 9 yr for boys. Less delay in the onset of treatment, longer duration of treatment, and lower chronological and bone age at the onset of treatment all lead to greater final height. All children with onset of pubertal symptoms before age 8 in girls and age 9 in boys should be evaluated for possible treatment. Treatment is appropriate in children with rapidly progressing puberty, accelerated bone maturation, and compromise of adult height prediction, regardless of bone age or chronological age at time of evaluation. However, once treatment is considered appropriate, it should be initiated quickly, because longer delays lead to shorter final height. In addition, the longer the treatment is continued, the greater is the final height outcome.
Human Pheochromocytomas Express Orexin Receptor Type 2 Gene and Display an in Vitro Secretory Response to Orexins A and B
Authors: Mazzocchi G. , Malendowicz L. K. , Aragona F. , Rebuffat P. , Gottardo L. , Nussdorfer G. G.
Published: January 2014

Orexins A and B are hypothalamic peptides, that act through two receptor subtypes, called OX1-R and OX2-R. OX1-R selectively binds orexin A, whereas OX2-R is nonselective for both orexins. High levels of OX1-R mRNA and low levels of OX2-R mRNA have been previously detected in the human adrenal cortex and medulla. Here we demonstrated by RT-PCR the expression of the OX2-R, but not the OX1-R, gene in 10 benign secreting pheochromocytomas. Both orexins A and B stimulated catecholamine secretion from pheochromocytoma slices; the maximal effective concentration was 10−8 mol/liter. Orexins A and B (10−8 mol/liter) increased IP3, but not cAMP production, by tumor slices, and the effect was blocked by the PLC inhibitor U-73122. The catecholamine response to 10−8 mol/liter orexins A and B was abolished by either U-73122 or the PKC antagonist calphostin C and was unaffected by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89. Collectively, these findings suggest that orexins stimulate catecholamine secretion from human pheochromocytomas, acting through OX2-R coupled to the PLC-PKC signaling pathway.
Up-Regulation by IGF-I of Proliferating Cell Nuclear Antigen and Bcl-2 Protein Expression in Human Uterine Leiomyoma Cells
Authors: Gao Zhijian , Matsuo Hiroya , Wang Yin , Nakago Satoshi , Maruo Takeshi
Published: January 2014

IGF-I has been reported to play a role in regulating proliferation of human leiomyoma cells. There is, however, little evidence to suggest that IGF-I inhibits apoptosis in the leiomyoma cells. The present study was conducted to elucidate whether IGF-I affects apoptosis and Bcl-2 protein expression, an apoptosis-inhibiting gene product, in cultured leiomyoma cells. In addition, we examined the effect of IGF-I on proliferating cell nuclear antigen (PCNA) expression in cultured leiomyoma cells. Isolated human leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% FBS for 120 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of graded concentrations of IGF-I (1.0, 10, and 100 ng/ml). The effects of IGF-I on Bcl-2 protein and PCNA expression in cultured leiomyoma cells were assessed by Western immunoblot analysis and immunocytochemical staining, whereas the effects of IGF-I on the cell viability and apoptosis of the cultured cells were determined by 3-(4,5-dimethylatriazol-2-yl)-2,5diphenyltetrasodium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick end labeling assay, respectively. Immunocytochemical staining demonstrated that IGF-I treatment resulted in the increase in PCNA labeling index in cultured leiomyoma cells in a dose-dependent manner. Immunoblot analysis of proteins extracted from the cultured leiomyoma cells revealed that the addition of IGF-I (10 and 100 ng/ml) significantly increased the expression of 35-kDa immunoreactive PCNA and 26-kDa Bcl-2 protein, compared with those in control cultures. Cell survival and proliferation of cultured leiomyoma cells, assessed by MTT assay, was significantly augmented by IGF-I treatment, compared with those of control cultures. Terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick end labeling assay showed that the apoptosis-positive rate of leiomyoma cells treated with IGF-I was significantly decreased, compared with that in control cultures. The present results suggest that IGF-I plays crucial roles in leiomyoma cell growth, not only in promoting the proliferative potential by up-regulation of PCNA expression but also in down-regulating apoptosis by up-regulation of Bcl-2 protein expression in leiomyoma cells.
Pretreatment with Recombinant Human TSH Changes the Regional Distribution of Radioiodine on Thyroid Scintigrams of Nodular Goiters
Authors: Nieuwlaat Willy-Anne , Hermus Ad R. , Sivro-Prndelj Ferida , Corstens Frans H. , Huysmans Dyde A.
Published: January 2014

In a recent study, we demonstrated that pretreatment with a single, low dose of recombinant human TSH (rhTSH) doubles 24-h thyroid radioactive iodine uptake in patients with nodular goiter. The purpose of the present study was to investigate whether rhTSH pretreatment induces changes in the regional distribution of radioiodine as visualized on thyroid scintigrams in these patients.

Anterior planar thyroid 123I scintigrams were obtained in 26 patients with a nodular goiter (23 women and 3 men; age, 62 ± 9 yr, mean ± sd; thyroid weight, 165 ± 72 g) 24 h after administration of a diagnostic dose of radioiodine. All patients were studied twice: first, without rhTSH pretreatment (baseline study), and second, after an im injection of 0.01 mg (n = 10) or 0.03 mg rhTSH (n = 16), given 24 h before radioiodine administration (rhTSH study). For quantification of regional differences in radioiodine uptake, a region of interest method was used.

Upon visual inspection, baseline scintigrams showed a heterogeneous uptake of radioiodine. In general, rhTSH scintigrams also showed heterogeneous radioiodine uptake. In some patients, the distribution of radioiodine in the rhTSH scintigram was considerably more homogeneous than in the baseline scintigram. In a few patients, originally“ cold” areas had changed into “hot” ones, whereas originally hot areas had changed into cold ones. Quantification of regional radioiodine uptake showed that pretreatment with rhTSH caused a larger increase in radioiodine uptake in relatively cold areas and a smaller increase in radioiodine uptake in relatively hot areas, compared with the increase in radioiodine uptake in the entire thyroid. In patients with a baseline serum TSH level of 0.5 mU/liter or lower, the increase in radioiodine uptake in relatively cold areas was significantly larger than in patients with a baseline serum TSH level higher than 0.5 mU/liter.

In conclusion, a single, low dose of rhTSH not only doubled 24-h radioactive iodine uptake but also caused a more homogeneous distribution of radioiodine within the thyroid gland in patients with a nodular goiter by stimulating radioiodine uptake in relatively cold areas more than in relatively hot areas. This was most marked in patients with a low baseline serum TSH level. Our data suggest that pretreatment with rhTSH may improve the efficacy of radioiodine treatment for volume reduction of nodular goiters, especially in patients with a low baseline serum TSH level.
Adipocytes Exhibit Abnormal Subcellular Distribution and Translocation of Vesicles Containing Glucose Transporter 4 and Insulin-Regulated Aminopeptidase in Type 2 Diabetes Mellitus: Implications Regarding Defects in Vesicle Trafficking
Authors: Maianu Lidia , Keller Susanna R. , Garvey W. Timothy
Published: January 2014

Insulin resistance in type 2 diabetes is due to impaired stimulation of the glucose transport system in muscle and fat. Different defects are operative in these two target tissues because glucose transporter 4 (GLUT 4) expression is normal in muscle but markedly reduced in fat. In muscle, GLUT 4 is redistributed to a dense membrane compartment, and insulin-mediated translocation to plasma membrane (PM) is impaired. Whether similar trafficking defects are operative in human fat is unknown. Therefore, we studied subcellular localization of GLUT4 and insulin-regulated aminopeptidase (IRAP; also referred to as vp165 or gp160), which is a constituent of GLUT4 vesicles and also translocates to PM in response to insulin. Subcutaneous fat was obtained from eight normoglycemic control subjects (body mass index, 29 ± 2 kg/m2) and eight type 2 diabetic patients (body mass index, 30 ± 1 kg/m2; fasting glucose, 14 ± 1 mm). In adipocytes isolated from diabetics, the basal 3-O-methylglucose transport rate was decreased by 50% compared with controls (7.1 ± 2.9 vs. 14.1 ± 3.7 mmol/mm2 surface area/min), and there was no increase in response to maximal insulin (7.9 ± 2.7 vs. 44.5 ± 9.2 in controls). In membrane subfractions from controls, insulin led to a marked increase of IRAP in the PM from 0.103 ± 0.04 to 1.00± 0.33 relative units/mg protein, concomitant with an 18% decrease in low-density microsomes and no change in high-density microsomes (HDM). In type 2 diabetes, IRAP overall expression in adipocytes was similar to that in controls; however, two abnormalities were observed. First, in basal cells, IRAP was redistributed away from low-density microsomes, and more IRAP was recovered in HDM (1.2-fold) and PM (4.4-fold) from diabetics compared with controls. Second, IRAP recruitment to PM by maximal insulin was markedly impaired. GLUT4 was depleted in all membrane subfractions (43–67%) in diabetes, and there was no increase in PM GLUT4 in response to insulin. Type 2 diabetes did not affect the fractionation of marker enzymes. We conclude that in human adipocytes: 1) IRAP is expressed and translocates to PM in response to insulin; 2) GLUT4 depletion involves all membrane subfractions in type 2 diabetes, although cellular levels of IRAP are normal; and 3) in type 2 diabetes, IRAP accumulates in membrane vesicles cofractionating with HDM and PM under basal conditions, and insulin-mediated recruitment to PM is impaired. Therefore, in type 2 diabetes, adipocytes express defects in trafficking of GLUT4/IRAP-containing vesicles similar to those causing insulin resistance in skeletal muscle.
Skeletal Muscle Lipid Content and Insulin Resistance: Evidence for a Paradox in Endurance-Trained Athletes
Authors: Goodpaster Bret H. , He Jing , Watkins Simon , Kelley David E.
Published: January 2014

We examined the hypothesis that an excess accumulation of intramuscular lipid (IMCL) is associated with insulin resistance and that this may be mediated by the oxidative capacity of muscle. Nine sedentary lean (L) and 11 obese (O) subjects, 8 obese subjects with type 2 diabetes mellitus (D), and 9 lean, exercise-trained (T) subjects volunteered for this study. Insulin sensitivity (M) determined during a hyperinsulinemic (40 mU·m−2min−1) euglycemic clamp was greater (P < 0.01) in L and T, compared with O and D (9.45 ± 0.59 and 10.26 ± 0.78 vs. 5.51 ± 0.61 and 1.15 ± 0.83 mg·min−1kg fat free mass−1, respectively). IMCL in percutaneous vastus lateralis biopsy specimens by quantitative image analysis of Oil Red O staining was approximately 2-fold higher in D than in L (3.04 ± 0.39 vs. 1.40 ± 0.28% area as lipid; P < 0.01). IMCL was also higher in T (2.36 ± 0.37), compared with L (P < 0.01). The oxidative capacity of muscle determined with succinate dehydrogenase staining of muscle fibers was higher in T, compared with L, O, and D (50.0 ± 4.4, 36.1 ± 4.4, 29.7 ± 3.8, and 33.4 ± 4.7 optical density units, respectively; P < 0.01). IMCL was negatively associated with M (r = −0.57, P < 0.05) when endurance-trained subjects were excluded from the analysis, and this association was independent of body mass index. However, the relationship between IMCL and M was not significant when trained individuals were included. There was a positive association between the oxidative capacity and M among nondiabetics (r = 0.37, P < 0.05). In summary, skeletal muscle of trained endurance athletes is markedly insulin sensitive and has a high oxidative capacity, despite having an elevated lipid content. In conclusion, the capacity for lipid oxidation may be an important mediator of the association between excess muscle lipid accumulation and insulin resistance.
The BB-Paraoxonase Genotype Is Associated with Impaired Brachial Reactivity after Acute Hypertriglyceridemia in Healthy Subjects
Authors: Paolisso Giuseppe , Manzella Daniela , Tagliamonte Maria Rosaria , Barbieri Michelangela , Marfella Raffaele , Zito Guido , Bonafè Massimiliano , Giugliano Dario , Franceschi Claudio , Varricchio Michele
Published: January 2014

The possible relationship between paraoxonase (PON) gene polymorphism and brachial reactivity in healthy adult subjects in the presence of acute hypertriglyceridemia (HT), as a prooxidant factor, was investigated. In 101 healthy subjects the response to flow- induced vasodilatation was measured before and after Intralipid infusion. In the same subjects the A/B PON polymorphism was detected. The frequency was 0.545 for AA genotype, 0.356 for the AB genotype, and 0.099 for the BB genotype. At baseline all genotype groups had a similar increase in brachial artery diameter and flow. After Intralipid infusion, subjects sharing the BB genotype had a significant decrease vs. baseline values in changes in brachial artery diameter (P for trend < 0.001 vs. the other genotypes), but not in flow. In a subgroup of 55 subjects distributed among the 3 PON genotypes the same study protocol was repeated by buccal nitroglycerine administration to study the endothelium-independent vasodilatation. Again, subjects with the BB genotype had the worse vasodilation (P for trend < 0.001). Furthermore, subjects sharing the BB genotype had the lowest endothelium-independent and -dependent changes in diameter (P for trend < 0.001 vs. the other genotypes) independently of gender ratio, basal plasma triglycerides concentrations, and changes in plasma triglycerides concentrations. In conclusion, our study demonstrates that transient HT decreases vascular reactivity more in subjects with the PON BB genotype than in those with the other PON genotypes.
Disruption of the Nocturnal Testosterone Rhythm by Sleep Fragmentation in Normal Men
Authors: Luboshitzky Rafael , Zabari Ziva , Shen-Orr Zilla , Herer Paula , Lavie Peretz
Published: January 2014

Recently, we have demonstrated that in normal men the nocturnal testosterone rise antedated the first rapid eye movement (REM) sleep episode by about 90 min and was correlated with REM latency. To further elucidate whether the diurnal testosterone rhythm is a sleep-related phenomenon or controlled by the circadian clock, we determined serum testosterone levels in 10 men during the ultrashort 7/13 sleep-wake cycle paradigm. Using this schedule, subjects experienced partial sleep deprivation and fragmented sleep for a 24-h period. Serum testosterone levels were determined every 20 min between 1900–0700 h with simultaneous sleep recordings during the 7-min sleep attempts. The results were compared with those obtained in men during continuous sleep. Although mean levels and area under the curve of testosterone were similar in both groups, fragmented sleep resulted in a significant delay in testosterone rise (03:24 h ± 1:13 vs. 22:35 h ± 0:22). During fragmented sleep, nocturnal testosterone rise was observed only in subjects who showed REM episodes (4/10). Our findings indicate that the sleep-related rise in serum testosterone levels is linked with the appearance of first REM sleep. Fragmented sleep disrupted the testosterone rhythm with a considerable attenuation of the nocturnal rise only in subjects who did not show REM sleep.
Ligands for Peroxisome Proliferator-Activated Receptor γ Inhibit Growth and Induce Apoptosis of Human Papillary Thyroid Carcinoma Cells
Authors: Ohta Kazuyasu , Endo Toyoshi , Haraguchi Kazutaka , Hershman Jerome M. , Onaya Toshimasa
Published: January 2014

Ligands for peroxisome proliferator-activated receptor γ (PPARγ) induce apoptosis and exert antiproliferative effects on several carcinoma cell lines. The present study investigates the expression of PPARγ and the possibility that agonists for PPARγ also inhibit the growth of human thyroid carcinoma cells. We examined this hypothesis using six cell lines, designated BHP thyroid carcinoma cells, which originated from patients with papillary thyroid carcinoma. RT-PCR analysis revealed that the thyroid carcinoma cell lines BHP2–7, 7–13, 10–3, and 18–21 express PPARγ. More PPARγ was expressed in carcinoma than in adjacent normal thyroid tissue in three of six samples of human papillary carcinoma of the thyroid.

PPARγ-positive thyroid carcinoma cells were treated with agonists of PPARγ, troglitazone, BRL 49653, and 15-deoxy-Δ12,14-prostaglandin J2. Troglitazone (10μ mol/L), BRL 49653 (10 μmol/L), and 15-deoxy-Δ12,14-prostaglandin J2 (1 μg/mL) decreased[ 3H]thymidine incorporation and reduced cell number, respectively, in BHP carcinoma cell lines that expressed PPARγ. Under low serum conditions, ligands for PPARγ induced condensation of the nucleus and fragmentation of chromatin into nucleosome ladders. These findings indicate that the death of thyroid carcinoma cells is a form of apoptosis.

To investigate the molecular mechanism of the apoptosis, we assessed expression of the apoptosis-regulatory genes bcl-2, bax, and c-myc. Troglitazone significantly increased the expression of c-myc messenger RNA but had no effect on the expression of bcl-2 and bax in thyroid carcinoma cells. These results suggest that, at least in part, the induction of apoptosis in human papillary thyroid carcinoma cells may be due to an increase of c-myc.

Troglitazone (500 mg/kg·day) significantly inhibited tumor growth and prevented distant metastasis of BHP18–21 tumors in nude mice in vivo.

Taken together, these results suggest that PPARγ agonist inhibit cell growth of some types of human thyroid cancer.
Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency: Alterations in Cortisol Pharmacokinetics at Puberty
Authors: Charmandari Evangelia , Hindmarsh Peter C. , Johnston Atholl , Brook Charles G. D.
Published: January 2014

In congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, treatment with glucocorticoid and mineralocorticoid substitution is not always satisfactory. Suboptimal control is often observed in pubertal patients, despite adequate replacement doses and adherence to treatment. We investigated whether the pubertal process is associated with alterations in cortisol pharmacokinetics resulting in a loss of control of the hypothalamic-pituitary-adrenal axis.

We determined the pharmacokinetics of hydrocortisone administered iv as a bolus. A dose of 15 mg/m2 body surface area was given to 14 prepubertal (median age, 9.4 yr; range, 6.1–10.8 yr), 20 pubertal (median, 13.5 yr; range, 10.6–16.8 yr), and 6 postpubertal (median, 18.2 yr; range, 17.2–20.3 yr) patients with salt-wasting CAH. All patients were on standard replacement therapy with hydrocortisone and 9α-fludrocortisone. Serum total cortisol concentrations were measured at 10-min intervals for 6 h following iv hydrocortisone bolus and analyzed using a solid-phase RIA.

The serum total cortisol clearance curve was monoexponential. Mean clearance was significantly higher in the pubertal group (mean, 427.0 mL/min; sd, 133.4) compared with the prepubertal (mean, 248.7 mL/min; sd, 100.6) and postpubertal (mean, 292.4 mL/min; sd, 106.3) (one-way ANOVA, F = 9.8, P < 0.001) groups. This effect persisted after adjustment for body mass index. The mean volume of distribution was also significantly higher in the pubertal (mean, 49.5 L; sd, 12.2) than the prepubertal (mean, 27.1 L; sd, 8.4) patients but not in the postpubertal (mean, 40.8 L; sd, 16) (ANOVA, F = 15.2, P < 0.001) patients. The significance remained after correction for body mass index. There was no significant difference in mean half-life of total cortisol in prepubertal (mean, 80.2 min; sd, 19.4), pubertal (mean, 84.4 min; sd, 24.9), and postpubertal (mean, 96.7 min; sd, 9.9) patients. Similar differences between groups were observed when the pharmacokinetic parameters of free cortisol were examined. In addition, the half-life of free cortisol was significantly shorter in females compared with males (P = 0.04).

These data suggest that puberty is associated with alterations in cortisol pharmacokinetics resulting in increased clearance and volume of distribution with no change in half-life. These alterations probably reflect changes in the endocrine milieu at puberty and may have implications for therapy of CAH and other conditions requiring cortisol substitution in the adolescent years.
Survival of Human Ovarian Follicles from Fetal to Adult Life: Apoptosis, Apoptosis-Related Proteins, and Transcription Factor GATA-41
Authors: Vaskivuo Tommi E. , Anttonen Mikko , Herva Riitta , Billig Håkan , Dorland Marinus , te Velde Egbert R. , Stenbäck Frej , Heikinheimo Markku , Tapanainen Juha S.
Published: January 2014

The majority of oocytes present in fetal ovaries are depleted before birth, and only about 400 will ovulate during the normal fertile life span. Studies on animals have shown that apoptosis is the mechanism behind oocyte depletion and follicular atresia. In the present study, we investigated the extent and localization of apoptosis in human fetal (aged 13–40 weeks) and adult ovaries. Furthermore, the expression of apoptosis-regulating proteins, bcl-2 and bax, and the relationship of transcription factor GATA-4 were studied. Apoptosis was found in ovarian follicles throughout fetal and adult life. During fetal development, apoptosis was localized mainly to primary oocytes and was highest between weeks 14–28, decreasing thereafter toward term. Expression of bcl-2 was observed only in the youngest fetal ovaries (weeks 13–14), and bax was present in the ovaries throughout the entire fetal period. In adult ovaries, apoptosis was detected in granulosa cells of secondary and antral follicles, and Bcl-2 and bax were expressed from primary follicles onwards. During fetal ovarian development, GATA-4 messenger RNA and protein were localized to the granulosa cells, with expression being highest in the youngest ovaries and decreasing somewhat toward term. The expression pattern of GATA-4 suggests that it may be involved in the mechanisms protecting granulosa cells from apoptosis from fetal to adult life. The results indicate that depletion of ovarian follicles in the human fetus occurs through intrinsic mechanisms of apoptosis in oocytes, and later in adult life the survival of growing follicles may be primarily determined by granulosa cell apoptosis.
Heterozygous Mutation in the Cholesterol Side Chain Cleavage Enzyme (P450scc) Gene in a Patient with 46,XY Sex Reversal and Adrenal Insufficiency
Authors: Tajima Toshihiro , Fujieda Kenji , Kouda Naoya , Nakae Jun , Miller Walter L.
Published: January 2014

Cytochrome P450scc, the mitochondrial cholesterol side chain cleavage enzyme, is the only enzyme that catalyzes the conversion of cholesterol to pregnenolone and, thus, is required for the biosynthesis of all steroid hormones. Congenital lipoid adrenal hyperplasia is a severe disorder of steroidogenesis in which cholesterol accumulates within steroidogenic cells and the synthesis of all adrenal and gonadal steroids is impaired, hormonally suggesting a disorder in P450scc. However, congenital lipoid adrenal hyperplasia is caused by mutations in the steroidogenic acute regulatory protein StAR; it has been thought that P450scc mutations are incompatible with human term gestation, because P450scc is needed for placental biosynthesis of progesterone, which is required to maintain pregnancy. In studying patients with congenital lipoid adrenal hyperplasia, we identified an individual with normal StAR and SF-1 genes and a heterozygous mutation in P450scc. The mutation was found in multiple cell types, but neither parent carried the mutation, suggesting it arose de novo during meiosis, before fertilization. The patient was atypical for congenital lipoid adrenal hyperplasia, having survived for 4 yr without hormonal replacement before experiencing life-threatening adrenal insufficiency. The P450scc mutation, an in-frame insertion of Gly and Asp between Asp271 and Val272, was inserted into a catalytically active fusion protein of the P450scc system (H2N-P450scc-Adrenodoxin Reductase-Adrenodoxin-COOH), completely inactivating enzymatic activity. Cotransfection of wild-type and mutant vectors showed that the mutation did not exert a dominant negative effect. Because P450scc is normally a slow and inefficient enzyme, we propose that P450scc haploinsufficiency results in subnormal responses to ACTH, so that recurrent ACTH stimulation leads to a slow accumulation of adrenal cholesterol, eventually causing cellular damage. Thus, although homozygous absence of P450scc should be incompatible with term gestation, haploinsufficiency of P450scc causes a late-onset form of congenital lipoid adrenal hyperplasia that can be explained by the same two-hit model that has been validated for congenital lipoid adrenal hyperplasia caused by StAR deficiency.
Mutational Analysis and Genotype-Phenotype Correlation of the PHEX Gene in X-Linked Hypophosphatemic Rickets
Authors: Holm Ingrid A. , Nelson Anne E. , Robinson Bruce G. , Mason Rebecca S. , Marsh Deborah J. , Cowell Christopher T. , Carpenter Thomas O.
Published: January 2014

PHEX is the gene defective in X-linked hypophosphatemic rickets. In this study, analysis of PHEX revealed mutations in 22 hypophosphatemic rickets patients, including 16 of 28 patients in whom all 22 PHEX exons were studied. In 13 patients, in whom no PHEX mutation had been previously detected in 17 exons, the remaining 5 PHEX exons were analyzed and mutations found in 6 patients. Twenty different mutations were identified, including 16 mutations predicted to truncate PHEX and 4 missense mutations.

Phenotype analysis was performed on 31 hypophosphatemic rickets patients with PHEX mutations, including the 22 patients identified in this study, 9 patients previously identified, and affected family members. No correlation was found between the severity of disease and the type or location of the mutation. However, among patients with a family history of hypophosphatemic rickets, there was a trend toward more severe skeletal disease in patients with truncating mutations. Family members in more recent generations had a milder phenotype. Postpubertal males had a more severe dental phenotype. In conclusion, although identifying mutations in PHEX may have limited prognostic value, genetic testing may be useful for the early identification and treatment of affected individuals. Furthermore, this study suggests that other genes and environmental factors affect the severity of hypophosphatemic rickets.
Chronic Insomnia Is Associated with Nyctohemeral Activation of the Hypothalamic-Pituitary-Adrenal Axis: Clinical Implications
Authors: Vgontzas Alexandros N. , Bixler Edward O. , Lin Hung-Mo , Prolo Paolo , Mastorakos George , Vela-Bueno Antonio , Kales Anthony , Chrousos George P.
Published: January 2014

Although insomnia is, by far, the most commonly encountered sleep disorder in medical practice, our knowledge in regard to its neurobiology and medical significance is limited. Activation of the hypothalamic-pituitary-adrenal axis leads to arousal and sleeplessness in animals and humans; however, there is a paucity of data regarding the activity of the hypothalamic-pituitary-adrenal axis in insomniacs. We hypothesized that chronic insomnia is associated with increased plasma levels of ACTH and cortisol. Eleven young insomniacs (6 men and 5 women) and 13 healthy controls (9 men and 4 women) without sleep disturbances, matched for age and body mass index, were monitored in the sleep laboratory for 4 consecutive nights, whereas serial 24-h plasma measures of ACTH and cortisol were obtained during the fourth day. Insomniacs, compared with controls, slept poorly (significantly higher sleep latency and wake during baseline nights). The 24-h ACTH and cortisol secretions were significantly higher in insomniacs, compared with normal controls (4.2 ± 0.3 vs. 3.3 ± 0.3 pm, P = 0.04; and 218.0 ± 11.0 vs. 190.4 ± 8.3 nm, P = 0.07). Within the 24-h period, the greatest elevations were observed in the evening and first half of the night. Also, insomniacs with a high degree of objective sleep disturbance (% sleep time < 70), compared with those with a low degree of sleep disturbance, secreted a higher amount of cortisol. Pulsatile analysis revealed a significantly higher number of peaks per 24 h in insomniacs than in controls (P < 0.05), whereas cosinor analysis showed no differences in the temporal pattern of ACTH or cortisol secretion between insomniacs and controls. We conclude that insomnia is associated with an overall increase of ACTH and cortisol secretion, which, however, retains a normal circadian pattern. These findings are consistent with a disorder of central nervous system hyperarousal rather than one of sleep loss, which is usually associated with no change or decrease in cortisol secretion or a circadian disturbance. Chronic activation of the hypothalamic-pituitary-adrenal axis in insomnia suggests that insomniacs are at risk not only for mental disorders, i.e. chronic anxiety and depression, but also for significant medical morbidity associated with such activation. The therapeutic goal in insomnia should be to decrease the overall level of physiologic and emotional arousal, and not just to improve the nighttime sleep.
Overexpression of CXC Chemokines by an Adrenocortical Carcinoma: A Novel Clinical Syndrome
Authors: Schteingart David E. , Giordano Thomas J. , Benitez Ricardo S. , Burdick Marie D. , Starkman Monica N. , Arenberg Douglas A. , Strieter Robert M.
Published: January 2014

A patient with adrenocortical carcinoma presented with fever, leukocytosis, and increased acute phase reactants. The tumor was infiltrated with neutrophils. Immunohistochemical staining of the tumor showed positive signal for epithelial neutrophil-activating protein-78, an angiogenic and chemotactic CXC chemokine. Conditioned medium from tumor-derived cells (RL-251) showed high concentration of IL-8, epithelial neutrophil-activating protein-78, Gro α, and Gro γ, angiogenic CXC chemokines with a potential role in tumorigenesis. An adrenal cancer/severe combined immunodeficiency mouse chimera was developed. Mice grew tumors rapidly, and circulating levels of IL-8 and epithelial neutrophil-activating protein-78 were detected. In contrast, animals transplanted with NCI-H295 cells, a nonchemokine-secreting cell line, grew tumors more slowly and did not have detectable chemokine levels. Similar to the patient, mice with RL-251 tumors developed marked leukocytosis and neutrophilia, and their tumors were infiltrated with neutrophils. Mice were passively immunized with epithelial neutrophil-activating protein-78 antisera. A marked decrease in tumor growth was observed. Potential for chemokine production by other adrenocortical tumors was investigated by RT-PCR in archival material. Six of seven adrenal carcinomas and one of three adenomas had cDNA for IL-8; six of seven carcinomas and the three adenomas had cDNA for epithelial neutrophil-activating protein-78. We concluded that the clinical presentation of this case resulted from increased tumor production of chemotactic chemokines. Through their angiogenic and chemotactic properties these chemokines may play an important role in adrenal tumorigenesis.
Metformin Does Not Adversely Affect Hormonal and Symptomatic Responses to Recurrent Hypoglycemia
Authors: Fruehwald-Schultes Bernd , Kern Werner , Oltmanns Kerstin M. , Sopke Stefan , Toschek Barbara , Born Jan , Fehm Horst L. , Peters Achim
Published: January 2014

Body weight gain and severe hypoglycemia are the major adverse effects of insulin therapy in type 2 diabetic patients. Metformin has been shown to prevent insulin therapy-induced body weight gain when used in combination with insulin. However, the effects of metformin on hormonal and symptomatic responses to hypoglycemia mediating hypoglycemia awareness have not been assessed to date. Fifteen young healthy men were treated with 850 mg metformin and placebo twice daily for a 16-d period in a double blind, cross-over design. On the last 2 d of the treatment period, the subjects underwent three hypoglycemic clamp experiments, with the first and the last performed with identical patterns of plasma glucose decrease. Differences between the effects of metformin and placebo (effect of metformin) as well as between first and last hypoglycemic clamps (effect of antecedent hypoglycemia) were assessed. Antecedent hypoglycemia significantly reduced epinephrine, ACTH, cortisol, glucagon, GH, and symptomatic responses to hypoglycemia (P < 0.05 for all variables). There was no detectable effect of metformin on epinephrine, norepinephrine, ACTH, cortisol, glucagon, or autonomic symptomatic response to hypoglycemia (P > 0.05 for all comparisons), except that metformin slightly increased the response of GH to hypoglycemia (P = 0.039). The latter finding may be due to an IGF-I-reducing effect of metformin, as after 14 d of metformin treatment baseline levels of IGF-I were significantly lower than in the placebo condition (236.9 ± 13.9 vs. 263.2 ± 14.4 μg/liter; P= 0.015). The data indicate that metformin does not adversely affect hormonal and symptomatic responses to hypoglycemia. This finding appears to be relevant with regard to the safety of the combination of metformin with insulin therapy.
Gender Differences in rhGH-Induced Changes in Body Composition in GH-Deficient Adults
Authors: Span Jan P. T. , Pieters Gerlach F. F. M. , Sweep Fred G. J. , Hermus Ad R. M. M. , Smals Anthony G. H.
Published: January 2014

In GH-deficient adults, rhGH has pronounced effects on total body water, fat free mass, and fat mass. Recently, we observed a gender difference in IGF-I responsivity to rhGH that was sex steroid dependent. The aim of the present study was to assess the effect of rhGH therapy on body composition parameters with due attention to the gender differences in biological responsiveness to rhGH. Forty-four women [36.9 ± 11.9 yr (mean ± sd)] and 33 men (37.2 ± 13.8 yr) with GH deficiency were studied every 6 months during 2 yr. The treatment goal was to achieve IGF-I levels within the age-adjusted normal range. Total body water, fat free mass, and fat mass were measured by bioimpedantiometry. To reach the treatment goal, the daily rhGH dose (IU/kg/d) had to be significantly higher in women than in men at all time intervals. During rhGH therapy, total body water and fat free mass increased significantly in both men and women (P ≤ 0.01 by ANOVA), but changes were more pronounced in men. Fat mass decreased during rhGH treatment and reached its nadir at 6 months, which was more pronounced in men than in women (P = 0.02 by ANOVA). After the initial decrease, fat mass increased again and reached baseline values after 2 yr of treatment. In both men and women, the total body water and fat free mass increases were closely related to the IGF-I increments (P < 0.001 by Pearson’s correlation test). The decrease in fat mass correlated significantly with the increase in IGF-I in men (r = −0.89, P < 0.001), not in women. Confirming our earlier data, IGF-I responsivity to rhGH was significantly higher in men than in women at all time intervals (P < 0.01 by ANOVA). Total body water and fat free mass responsivities were also higher in men than in women (P < 0.01 by ANOVA). In conclusion, gender differences in IGF-I responsivities to rhGH are accompanied by gender differences in the extent of body composition changes to rhGH. Probably because of these gender differences in IGF-I responsivity, the increases of total body water and fat free mass to rhGH replacement were greater in men than in women. Remarkably, however, in men, only total body water and fat free mass responses relative to changes in IGF-I increased during the 2 yr of rhGH therapy (P= 0.02 and 0.01, respectively, by ANOVA). In our opinion, this phenomenon might be explained by the increasing target organ sensitivity to IGF-I over time.