bluuebunny/crossref_metadata_2025 huggingface dataset:
Short-Term Fasting Selectively Suppresses Leptin Pulse Mass and 24-Hour Rhythmic Leptin Release in Healthy Midluteal Phase Women without Disturbing Leptin Pulse Frequency or Its Entropy Control (Pattern Orderliness)1
Authors: Bergendahl M. , Iranmanesh A. , Evans W. S. , Veldhuis J. D.
Published: January 2014

Nutritional signals strongly regulate neuroendocrine axes, such as those subserving release of LH, GH, and TSH, presumptively in part via the adipocyte-derived neuroactive peptide leptin. In turn, leptin release is controlled by both acute (fasting) and long-term (adipose store) nutrient status. Here, we investigate the neuroendocrine impact of short-term (2.5-day) fasting on leptin release in healthy young women studied in the steroid-replete midluteal phase of the normal menstrual cycle. Eight women each underwent 24-h blood sampling at 10-min intervals during a randomly ordered 2.5-day fasting vs. fed session in separate menstrual cycles. Pulsatile leptin release was quantified by model-free Cluster analysis, the orderliness of leptin patterns by the approximate entropy statistic, and nyctohemeral leptin rhythmicity by cosinor analysis. Mean (24-h) serum leptin concentrations fell by 4.6-fold during fasting; namely, from 15.2 ± 2.3 to 3.4 ± 0.6 μg/L (P= 0.0007). Cluster analysis identified 13.9 ± 1.1 and 14.3± 1.1 leptin peaks per 24 h in the fed and fasting states (P = NS), and unchanging leptin interpeak intervals (89 ± 5.4 vs. 92 ± 5.3 min). Leptin peak area declined by 4.2-fold (155 ± 21 vs. 37 ± 7 area units, P = 0.004), due to a reduction in incremental leptin pulse amplitude (4.4 ± 0.7 vs. 1.0 ± 0.13 μg/L, P = 0.0011). The cosine amplitude and mesor (mean) of the 24-h leptin rhythm decreased by 4-fold, whereas the acrophase (timing of the nyctohemeral leptin peak) remained fixed. The approximate entropy of leptin release was stable, thus indicating preserved orderliness of leptin release patterns in fasting. Cross-correlation analysis revealed both positive (fed) and negative (fasting) leptin-GH relationships, but no leptin-LH correlations.

In summary, short-term (2.5-day) fasting profoundly suppresses 24-h serum leptin concentrations and pulsatile leptin release in the sex steroid-sufficient midluteal phase of healthy women via mechanisms that selectively attenuate leptin pulse area and incremental amplitude. In contrast, the pulse-generating, nyctohemeral phase-determining, and entropy-control mechanisms that govern 24-h leptin release are not altered by acute nutrient restriction at this menstrual phase. Leptin-GH (but not leptin-LH) showed nutrient-dependent positive (fed) and negative (fasting) cross-correlations. Whether similar neuroendocrine mechanisms supervise altered leptin signaling during short-term nutrient restriction in men, children, or postmenopausal women is not known.
3β-Hydroxysteroid Dehydrogenase/Δ5→4-Isomerase Activity Associated with the Human 17β-Hydroxysteroid Dehydrogenase Type 2 Isoform1
Authors: Suzuki Takashi , Sasano Hironobu , Andersson Stefan , Mason J. Ian
Published: January 2014

The type 2 isoform of human 17β-hydroxysteroid dehydrogenase (17βHSD2) efficiently catalyzes the oxidative metabolism of androgens and estrogens, and it is expressed in a large series of human peripheral tissues. To obtain a better understanding of the regulation of local steroid biosynthesis and metabolism in human tissues, we have established a dual steroidogenic activity of the 17βHSD2 enzyme after transfection of human 17βHSD2-transfected human embryonic kidney (293) cells. After transient transfection, the metabolism of testosterone, pregnenolone, and dehydroepiandrosterone (DHEA) in intact transfected 293 cells was evaluated by TLC-based radiometric assays. 17βHSD2-transfected cells converted 91% of testosterone (1 μmol/L) into androstenedione in a 2-h incubation period. In addition, pregnenolone (1 μmol/L) was converted to progesterone (18.5%), whereas DHEA (1 μmol/L) was metabolized to androstenedione (8.3% conversion) in a 15-h incubation period. The kinetics of the 3β-hydroxysteroid dehydrogenase (3βHSD) and 17βHSD2 activities using cell homogenate protein of stably transfected 293 cells indicated that the catalytic efficiency (apparent catalytic efficiency = maximum velocity/Km) of this 3βHSD activity is approximately 2000-fold (pregnenolone as substrate) or 3000-fold (DHEA as substrate) weaker than that of 17βHSD2 activity. It is noteworthy, however, that the apparent catalytic efficiency of the HSD3B2 gene product is only approximately 50-fold higher than that of the 3βHSD aspect of the 17βHSD2 gene product. Pregnenolone or DHEA effectively inhibited 17βHSD2 activity in a noncompetitive fashion. Furthermore, the potent 5α-reductase/3βHSD inhibitor, 17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5α-androstane-3-one, inhibited neither 3βHSD nor 17βHSD2 activities. We conclude that human 17βHSD2 enzyme exhibits 3βHSD activity. Notwithstanding that this 3βHSD activity is reduced compared to 17βHSD oxidative activity, it may account for at least some of the reports of 3βHSD activity found in human peripheral tissues that express notable amounts of the 17βHSD2 isozyme as well as in individuals with severe classic 3βHSD deficiency.
Atorvastatin Improves Postprandial Lipoprotein Metabolism in Normolipidemic Subjects1
Authors: Parhofer Klaus G. , Barrett P. Hugh R. , Schwandt Peter
Published: January 2014

Atorvastatin is a potent HMG-CoA reductase inhibitor that decreases low-density lipoprotein (LDL) cholesterol and fasting triglyceride concentrations. Because of the positive association between elevated postprandial lipoproteins and atherosclerosis, we investigated the effect of atorvastatin on postprandial lipoprotein metabolism. The effect of 4 weeks of atorvastatin therapy (10 mg/day) was evaluated in 10 normolipidemic men (30 ± 2 yr; body mass index, 22 ± 3 kg/m2; cholesterol, 4.84 ± 0.54 mmol/L; triglyceride, 1.47 ± 0.50 mmol/L; high-density lipoprotein cholesterol, 1.17 ± 0.18 mmol/L; LDL-cholesterol, 3.00 ± 0.49 mmol/L). Postprandial lipoprotein metabolism was evaluated with a standardized fat load (1300 kcal, 87% fat, 7% carbohydrates, 6% protein, 80,000 IU vitamin A) given after 12 h fast. Plasma was obtained every 2 h for 14 h. A chylomicron (CM) and a chylomicron-remnant (CR) fraction was isolated by ultracentrifugation, and triglycerides, cholesterol, apolipoprotein B, apoB-48, and retinyl-palmitate were determined in plasma and in each lipoprotein fraction. Atorvastatin therapy significantly (P < 0.001) decreased fasting cholesterol (−28%), triglycerides (−30%), LDL- cholesterol (-41%), and apolipoprotein B (−39%), whereas high-density lipoprotein cholesterol increased (4%, not significant). The area under the curve for plasma triglycerides (−27%) and CR triglycerides (−40%), cholesterol (−49%), and apoB-48 (−43%) decreased significantly (P < 0.05), whereas CR retinyl-palmitate decreased (−34%) with borderline significance (P = 0.08). However, none of the CM parameters changed with atorvastatin therapy. This indicates that, in addition to improving fasting lipoprotein concentrations, atorvastatin improves postprandial lipoprotein metabolism presumably by increasing CR clearance or by decreasing the conversion of CMs to CRs, thus increasing the direct removal of CMs from plasma.
Persistent Increase in Bone Turnover in Graves’ Patients with Subclinical Hyperthyroidism
Authors: Kumeda Yasuro , Inaba Masaaki , Tahara Hideki , Kurioka Yasuko , Ishikawa Tetsuro , Morii Hirotoshi , Nishizawa Yoshiki
Published: January 2014

Hyperthyroid patients exhibit accelerated bone loss by increased bone turnover, and normalization of thyroid function is associated with a significant attenuation of increased bone turnover, followed by an increase in bone mineral density. However, of patients with Graves’ disease (GD) maintained on antithyroid drug (ATD) treatment, some exhibit persistent suppression of TSH long after normalization of their serum free T3 (FT3) and free T4 (FT4) levels. The aim of this study was to examine whether bone metabolism is still enhanced in TSH-suppressed premenopausal GD patients with normal FT3 and FT4 levels after ATD therapy (n = 19) compared with that in TSH-normal premenopausal GD patients (n = 30), and to evaluate the relationship between serum TSH receptor antibody (TRAb), an indicator of disease activity of GD, and various biochemical markers of bone metabolism. No difference was found between the two groups in serum Ca, phosphorus, or intact PTH, or in urinary Ca excretion. Serum bone alkaline phosphatase (B-ALP), bone formation markers, and urinary excretions of pyridinoline (U-PYD) and deoxypyridinoline (U-DPD), which are bone resorption markers, were significantly higher in the TSH-suppression group than in the TSH-normal group (B-ALP, P < 0.05; U-PYD, P < 0.001; U-DPD, P < 0.001). For the group of all GD patients enrolled in this study, TSH, but neither FT3 nor FT4, exhibited a significant negative correlation with B-ALP (r = −0.300; P < 0.05), U-PYD (r= −0.389; P < 0.05), and U-DPD (r = −0.446; P < 0.05), whereas TRAb exhibited a highly positive and significant correlation with B-ALP (r = 0.566; P < 0.0001), U-PYD (r = 0.491; P < 0.001), and U-DPD (r = 0.549; P < 0.0001). Even in GD patients with normal TSH, serum TRAb was positively correlated with B-ALP (r = 0.638; P < 0.001), U-PYD (r = 0.638; P < 0.001), and U-DPD (r = 0.641; P < 0.001). In conclusion, it is important to achieve normal TSH levels during ATD therapy to normalize bone turnover. TRAb was not only a useful marker for GD activity, but was also a very sensitive marker for bone metabolism in GD patients during ATD treatment.
No Evidence for Linkage at Candidate Type 2 Diabetes Susceptibility Loci on Chromosomes 12 and 20 in United Kingdom Caucasians
Authors: Frayling Timothy M. , McCarthy Mark I. , Walker Mark , Levy Jonathan C. , O’Rahilly Stephen , Hitman Graham A. , Subba Rao Pamidighantam V. , Bennett Amanda J. , Jones Elizabeth C. , Menzel Stephan , Ellard Sian , Hattersley Andrew T.
Published: January 2014

Several studies have identified evidence for linkage between type 2 diabetes and the regions on chromosomes 12 and 20 containing the maturity-onset diabetes of the young (MODY) genes, hepatocyte nuclear factor-1α (HNF-1α) and HNF-4α. Two studies examining the HNF-1α region have demonstrated evidence for linkage at genome-wide levels of significance, whereas four studies examining the HNF-4α locus have resulted in evidence for linkage at more suggestive levels of significance. The demonstration of linkage to these regions in additional patient series will strengthen the evidence that susceptibility alleles exist at these loci. We therefore assessed the evidence for linkage to these regions using a large cohort of United Kingdom Caucasian type 2 diabetes-affected sibling pairs.

A maximum total of 315 affected full sibling pairs were typed for microsatellite markers across the MODY regions and, in a subset of families, for markers spanning the whole of chromosome 20. Evidence for linkage was assessed using a multipoint, mode of inheritance-free method. Linkage analysis did not reveal any significant evidence for excess allele sharing at any of the regions studied. Loci contributing sibling recurrence risks, relative to the general population risk, of 1.75 and 1.25 could be excluded for the HNF-1α and HNF-4α regions, respectively.

We have not confirmed in United Kingdom Caucasians the evidence for linkage previously reported on 12q and 20q. Our results highlight further the problems of replicating previous positive linkage results across different ethnic groups.
Estrogen Receptor Isoform Gene Expression in Ovarian Stromal and Epithelial Tumors*
Authors: Chu Simon , Mamers Pam , Burger Henry G. , Fuller Peter J.
Published: January 2014

The factors involved in the pathogenesis of ovarian cancers remain unclear, and the response of these tumors to hormonal therapy is limited. The identification of a second estrogen receptor gene (ERβ), expressed predominantly in ovarian granulosa cells, led us to explore its possible role in ovarian cancer, particularly in granulosa cell tumors (GCT). Several isoforms of ERβ have been identified. We sought to define the patterns of both ERα and ERβ gene expression in a panel of ovarian tumors consisting of GCT and serous and mucinous cystadenocarcinomas as well as in normal ovary. Expression was determined by RT-PCR using gene- and isoform-specific primers and probes combined with Southern blot analysis of the PCR products. Widespread expression of ERα was observed in all tumor types, but at relatively low levels. ERβ is expressed predominantly in GCT, with lower levels in mucinous tumors and very low levels in serous tumors. The ERβ2 splice variant previously reported in rodents was not observed. Only very low levels of the exon 5, exon 6, and exon 5/6 deletion variants were detected. The C-terminal truncation variant ERβcx, however, exhibited widespread expression across all the tumor types. As ERβcx has been shown to be a ligand-independent antagonist of ERα action, the relative ratios of ERβcx, ERα, and ERβ may influence the response of a tumor to antiestrogen therapy.
Significance of Pro12Ala Mutation in Peroxisome Proliferator-Activated Receptor-γ2 in Korean Diabetic and Obese Subjects*
Authors: Oh Eun Young , Min Kyeong Min , Chung Jae Hoon , Min Yong-Ki , Lee Myung-Shik , Kim Kwang-Won , Lee Moon-Kyu
Published: January 2014

Peroxisome proliferator-activated receptors (PPARs) are a nuclear hormone receptor superfamily of ligand-activated transcription factors, and the PPARγ subtype regulates adipocyte differentiation, lipid metabolism, and insulin sensitivity. There have been several reports on the relationship between the PPARγ2 Pro12Ala genotype and obesity or diabetes in Caucasians. The objective of this study was to examine the relationship between this mutation and obesity or diabetes in Korean subjects. Two hundred and twenty-nine Korean subjects, including 111 obese subjects (body mass index, >25 kg/m2) were included in this study. One hundred and eleven subjects had normal glucose tolerance, 60 had impaired glucose tolerance, and 58 had diabetes mellitus. We evaluated these subjects for the Pro12Ala mutation in the PPARγ gene using PCR-restriction fragment length polymorphism. Allele frequencies of the Pro12Ala missense mutation of PPARγ2 were not different among Korean subjects with normal glucose tolerance (qAla = 0.045), those with impaired glucose tolerance (qAla = 0.033), and those with diabetes mellitus (qAla = 0.043; P> 0.05). Allele frequencies of PPARγ2 Ala in obese subjects (qAla = 0.036) were not significantly different from those in nonobese subjects (qAla = 0.047). These results suggest that the Pro12Ala mutation in PPARγ is not associated with either diabetes or obesity and may not be an important determinant of obesity or diabetes in Korean subjects.
Spatio-Temporal Expression of the Trans-Acting Splicing Factors SF2/ASF and Heterogeneous Ribonuclear Proteins A1/A1B in the Myometrium of the Pregnant Human Uterus: A Molecular Mechanism for Regulating Regional Protein Isoform Expression in Vivo*
Authors: Pollard Alison J. , Sparey Colette , Robson Stephen C. , Krainer Adrian R. , Europe-Finner G. Nicholas
Published: January 2014

Many of the human myometrial proteins associated with uterine quiescence and the switch to coordinated contractions at the onset of labor exist as alternatively spliced isoforms. There is now extensive evidence to indicate that the nuclear concentrations of the trans-acting splicing regulators SF2/ASF and hnRNP A1/A1B are fundamental in regulating the expression of specific protein isoforms derived from alternative splicing of single precursor messenger ribonucleic acid transcripts. The question thus arose as to whether these factors were also involved in regulating the expression of specific myometrial protein species within different uterine regions during human gestation and parturition. SF2/ASF and hnRNP A1/A1B expression was therefore determined in paired upper (corpus) and lower segment myometrial samples taken from individual women at term/during spontaneous labor and compared with nonpregnant control samples using specific monoclonal antibodies. We report that SF2/ASF levels were substantially increased in the lower uterine region, and this was associated with a parallel decrease in levels of hnRNP A1/A1B during gestation. Conversely, the opposite pattern was observed within the upper uterine region during pregnancy, where hnRNP A1/A1B was significantly up-regulated and SF2/ASF levels were much less than those found in the lower uterine segment. The differential expression of hnRNP A1/A1B and SF2/ASF in the upper and lower uterine segments may have a primary role in defining the formation of specific myometrial protein species associated with the known contractile and relaxatory properties of these regions before and during parturition.
Preserved Male Fertility Despite Decreased Androgen Sensitivity Caused by a Mutation in the Ligand-Binding Domain of the Androgen Receptor Gene1
Authors: Giwercman Aleksander , Kledal Thomas , Schwartz Marianne , Giwercman Yvonne Lundberg , Leffers Henrik , Zazzi Henric , Wedell Anna , Skakkebæk Niels E.
Published: January 2014

Mutations in the androgen receptor gene are considered as incompatible with preservation of fertility and have been suggested as a cause of male infertility.

Two adult brothers, referred because of gynecomastia and hormonal levels in serum indicating androgen insensitivity (high sex hormone-binding globulin, and LH levels, despite extremely high testosterone concentration), turned out to be relatives to a third young man, referred independently of the two others and exhibiting identical clinical and hormonal stigmata. In all three men, we found a C→A substitution at position 2470 (exon 7) in the androgen receptor gene, leading to a Gln824Lys mutation in the ligand-binding domain of the receptor. Exploring the family history revealed that their grandfathers, on their mothers’ side, were brothers; and the Gln824Lys mutation was also found in the one of them who was still alive.

Binding studies with the mutant receptor in transfected COS-7 cells, with mibolerone as ligand, exhibited equal Kd (0.7 vs. 1.0 nmol/L), IC50 (0.8 vs. 1.1 nmol/L), and maximum binding (7.1 vs. 8.9 fmol/106 cells), as compared with the wild-type (WT) receptor. In a chloramphenicol acetyl transferase trans-activation assay, the activity of the mutant receptor was identical to that of the WT, when the synthetic androgen R1881 was used as a ligand; but with dihydrotestosterone, in concentrations up to 10 nmol/L, the activity of Gln824Lys mutated receptor was 10–62% of the WT variant.

Thus, Gln824Lys mutation was found, both in vivo and in vitro, to cause slight impairment of receptor function but was compatible with preservation of male fertility. The patients inherited the mutation from their grandfathers through their mothers, and one of the young men possessing the mutation has fathered a daughter.
Expression of Menin in Parathyroid Tumors
Authors: Bhuiyan M. M. R. , Sato M. , Murao K. , Imachi H. , Namihira H. , Takahara J.
Published: January 2014

The multiple endocrine neoplasia type 1 (MEN1) gene seems to be a tumor suppressor that encodes a 610-amino acid protein termed menin and that plays an important role in the development of MEN1 syndrome. Recent reports indicate that heterozygous germline mutations of this gene are responsible for the disease onset of MEN1. In this study we examined the expression of menin in parathyroid tumors from primary hyperparathyroidism (PHP), secondary hyperparathyroidism (SHP), and MEN1 and thyroid tumors including Basedow’s disease, thyroid cancer, and adrenocortical tumors. Both ribonucleic acid and protein from these tumors were applied to RT-PCR and Western blotting, respectively. Primers for RT-PCR were designed to amplify the sequence between exons 2 and 3 of the MEN1 gene. Specific antibody against menin was generated in guinea pigs immunized with the recombinant peptide from amino acid residues 443–535 of menin made by using glutathione-S-transferase (GST) gene fusion. Menin messenger ribonucleic acid was strongly expressed on RT-PCR analysis in the parathyroid tumors from both PHP and SHP. Western blotting revealed a specific band of approximately 67 kDa in parathyroid tumors from PHP and SHP, with a much weaker such band detected in thyroid tumors. Menin expression was down-regulated in MEN1 samples, including nonsense mutation and deletion mutant. These findings suggest that menin is predominantly synthesized and stored in parathyroid tumors resulting from PHP and SHP.
Thyroid Hormone Receptor Gene Expression in First Trimester Human Fetal Brain
Authors: Iskaros Joseph , Pickard Mark , Evans Ian , Sinha Arun , Hardiman Paul , Ekins Roger
Published: January 2014

Maternal thyroid hormone is transferred to the fetus early in pregnancy and is postulated to regulate brain development. Thyroid hormone nuclear receptor (TR) proteins are present in fetal brain, but their isoformal composition is unknown. We therefore investigated the ontogeny of TR isoforms and related splice variants in first trimester human fetal brain (n = 9) by semi-quantitative reverse transcriptase-polymerase chain reaction analysis. Expression of the TRβ1, TRα1 and c-erbAα2 isoforms was detected from 8.1 weeks gestation (wg). An additional truncated species was detected with the c-erbAα2 primer set, consistent with the c-erbAα3 splice variant previously described in the rat. All c-erbAα-derived transcripts were co-ordinately expressed and increased (ca.8-fold) between 8.1 and 13.9 wg. A more complex ontogenic pattern was observed for TRβ1, suggestive of a nadir between 8.4 and 12.0 wg. These findings point to an important role for the TRα1 isoform in mediating maternal thyroid hormone action during first trimester human fetal brain development.
Distribution and Abundance of Messenger Ribonucleic Acid for Growth Hormone Receptor Isoforms in Human Tissues1
Authors: Ballesteros Mercedes , Leung Kin-Chuen , Ross Richard J. M. , Iismaa Tiina P. , Ho Ken K. Y.
Published: January 2014

Two alternatively spliced exon 9 variants of human GH receptor (GHR) messenger ribonucleic acid (mRNA), GHR-(1–279) and GHR-(1–277), were recently identified in liver. They encode receptor proteins lacking most of the intracellular domain and inhibit GH action in a dominant negative manner. Little is known about tissue distribution and abundance of these GHR isoforms. We have developed quantitative RT-PCR assays specific for the full-length and truncated GHRs and investigated their expression in various human tissues and cell lines.

The mRNA of full-length GHR and GHR-(1–279) were readily detectable in all tissues investigated, with liver, fat, muscle, and kidney showing high levels of expression. These two receptor isoforms were also detected in a range of human cell lines, with strongest expression in IM9, a lymphoblastoid cell line. In contrast, GHR-(1–277) message was expressed at low levels in liver, fat, muscle, kidney, and prostate and in trace amount in IM9 cells.

Full-length GHR was the most abundant isoform, accounting for over 90% of total receptor transcripts in liver, fat, and muscle for quantitative RT-PCR. However, liver had 2- to 4-fold more full-length receptor mRNA and 16- to 40-fold more GHR-(1–277) mRNA than fat and muscle, whereas the mRNA levels of GHR-(1–279) were similar in the three tissues. GHR-(1–279) constituted less than 4% in liver and 7–10% in fat and muscle. GHR-(1–277) accounted for 0.5% of total GHR transcripts in liver and less than 0.1% in the other two tissues. These data suggest that the absolute and relative abundance of mRNA of the three GHR isoforms may be tissue specific. The regulation of expression of exon 9 alternatively spliced GHR variants may provide a potential mechanism for modulation of GH sensitivity at the tissue level.
Circulating Insulin-Like Growth Factor II and Colorectal Adenomas*
Authors: Renehan Andrew G. , Painter John E. , O’Halloran Domhnall , Atkin Wendy S. , Potten Christopher S. , O’Dwyer Sarah T. , Shalet Stephen M.
Published: January 2014

Circulating insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) may be risk factors for the development of colorectal cancer. On the other hand, IGF-II and IGFBP-2 are overexpressed in colorectal carcinomas. These contrasting backgrounds led us to investigate the relationship between serum IGF-I, IGF-II, IGFBP-2, and IGFBP-3 and the presence of colorectal adenomas, known precursors of colorectal carcinoma, in 345 volunteers attending a screening flexible sigmoidoscopy trial (entry criteria: healthy, aged 55–64 yr). The most striking finding was an elevated mean serum IGF-II in individuals with adenomas (n = 52) compared with controls (mean difference, 139 ng/mL; 95% confidence intervals, 82, 196; P < 0.0001). Logistic regression adjusting for confounding factors confirmed the significant association between IGF-II and adenoma occurrence (P < 0.0001) and revealed an additional positive association with serum IGFBP-2 (P < 0.0001). However, there was no association found between either serum IGF-I and/or IGFBP-3 and the presence of adenomas. Additionally, in 31 individuals with adenomas in whom levels were determined pre- and postpolypectomy, there was a significant fall in mean IGF-II (P < 0.001) and IGFBP-2 (P < 0.001) after adenoma removal, but no difference in IGF-II and IGFBP-2 concentrations between repeated samples in 20 individuals without adenomas. Immunohistochemical studies demonstrated IGF-II expression in 83% of all adenomas, which contrasted with absent expression in normal colonic expression and hyperplastic polyps. This study has shown for the first time that serum IGF-II may be a tumor marker in individuals with colorectal adenomas. Further studies are needed to validate these relationships in larger populations, including individuals undergoing colonoscopy.
Effects of Metyrapone Administration on Thyrotropin Secretion in Healthy Subjects–A Clinical Research Center Study*
Authors: Samuels M. H.
Published: January 2014

Although pharmacological doses of glucocorticoids suppress TSH secretion, less is known regarding the effects of physiological variations in cortisol levels on TSH. To study this issue, 12 healthy subjects each underwent 2 studies, in random order: 1) each subject received an infusion of saline for 48 h; and 2) each subject received an infusion of saline and oral administration of metyrapone (500 mg every 4 h) for 48 h. Cortisol and TSH levels were measured every 15 min during the final 24 h of each study, and resulting mean hormone levels during the 24-h periods were compared between the two studies. Metyrapone administration reduced serum cortisol levels by 39% between 0800 and 1345 h and by 47% between 0200 and 0745 h, with no significant changes during other time periods. Metyrapone increased daytime (0800–1945 h) mean TSH levels by 35%, with no change in nocturnal (2000–0745 h) TSH levels. This led to equalization of daytime and nocturnal TSH levels and abolition of the usual circadian variation in TSH. TSH pulse frequency was no different between the two studies, whereas daytime TSH pulse amplitude increased 33% during metyrapone administration. There were no changes in TSH responses to TRH, or in serum T3 or free T4 levels, at the end of the studies. These results suggest that the early morning increase in endogenous cortisol levels in healthy subjects causes the daytime decrease in TSH levels. In addition, these results show that very mild changes in cortisol levels within the physiological range are sufficient to affect TSH secretion.
The Decrease in Luteinizing Hormone Secretion in Response to Weight Reduction Is Inversely Related to the Severity of Insulin Resistance in Overweight Women*
Authors: Bützow Tarja L. , Lehtovirta Mikko , Siegberg Rita , Hovatta Outi , Koistinen Riitta , Seppälä Markku , Apter Dan
Published: January 2014

Controversial effects of weight reduction on gonadotropin secretion in obesity have been reported. As a result of pulsatility, single serum samples or frequent sampling studies are somewhat limited with regard to monitoring LH and FSH concentrations. We studied follicular phase nocturnal urinary (nu) LH and FSH secretion and glucose metabolism (150-min euglycemic hyperinsulinemic clamp) during 1 menstrual cycle/30-day period before and after weight reduction in 10 severely overweight infertility patients (age, 29 ± 3.1 yr; body mass index, 37.1 ± 3.3 kg/m2; ±sem). A 6-week very low calorie diet was followed by a 4-week normocaloric period. The urinary LH and FSH results reported represent samples taken 12 to 2 days before the LH surge, or 10 consecutive samples in the case of amenorrhea.

We observed a decrease of 8% (P < 0.001) in percent body fat mass and a 5% (P < 0.005) reduction in waist to hip ratio. Mean nu-LH decreased by 45%[ 6.06 ± 1.05 (±sem) to 3.22 ± 0.71 IU/L], whereas mean nu-FSH remained unchanged. Insulin-stimulated glucose uptake increased by 41% (P < 0.01), which was accounted for by a significant increase in nonoxidative glucose disposal (P = 0.003). Serum sex hormone-binding globulin concentrations increased by 39% (P < 0.01), and insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) levels increased by 46% (P < 0.05). Fasting serum insulin concentrations decreased by 38%, those of leptin by 37%, those of androstenedione by 32%, those of testosterone by 20% (all P < 0.01), and those of dehydroepiandrosterone sulfate by 13% (P < 0.05). The percent change in nu-LH correlated negatively with glucose uptake (r = −0.76; P < 0.01) and the increase in serum sex hormone-binding globulin (r = −0.85; P < 0.005) and positively with the percent change in waist to hip ratio (r = 0.79; P < 0.01). The absolute nu-LH levels after weight reduction correlated significantly with fasting insulin concentrations (r = 0.88; P < 0.001) and negatively with glucose uptake (r = −0.67; P < 0.05). No significant relationships were found between absolute levels or changes in nu-LH concentrations and leptin, IGF-I, IGFBP-3, or IGFBP-1 concentrations. Our findings suggest that weight reduction with a very low calorie diet results in a decrease in nu-LH concentrations, a reduction in the LH/FSH ratio, and FSH predominance favoring folliculogenesis. The decrease in LH concentrations is inversely related to the severity of insulin resistance. It is possible that the decrease in LH secretion with weight reduction is more dependent on the absolute levels of insulin sensitivity than on the degree of general adiposity.
Heterogeneity of Nuclear Lamin A Mutations in Dunnigan-Type Familial Partial Lipodystrophy*
Authors: Hegele Robert A. , Cao Henian , Anderson Carol M. , Hramiak Irene M.
Published: January 2014

We previously identified a novel mutation, namely LMNA R482Q, that was found to underlie Dunnigan-type partial lipodystrophy (FPLD) and diabetes in an extended Canadian kindred. We have since sequenced LMNA in five additional Canadian FPLD probands and herein report three new rare missense mutations in LMNA: V440M, R482W, and R584H. One severely affected subject was a compound heterozygote for both V440M and R482Q. The findings indicated that 1) a spectrum of LMNA mutations underlies FPLD; 2) aberrant lamin A, and not lamin C, is likely to underlie FPLD, as R584H occurs within LMNA sequence that is specific for lamin A; 3) the V440M mutation may not cause lipodystrophy on its own; 4) compound heterozygosity for V440M and R482Q is associated with a relatively more severe FPLD phenotype, but not with complete lipodystrophy; and 5) variation in the severity of the phenotype might be related to environmental factors.
Androgen Receptor Expression in Sertoli Cells as a Function of Seminiferous Tubule Maturation in the Human Cryptorchid Testis1
Authors: Regadera Javier , MartÍnez-GarcÍa Francisco , González-Peramato Pilar , Serrano Alvaro , Nistal Manuel , Suárez-Quian Carlos
Published: January 2014

Androgen receptor (AR) immunohistochemistry was performed in an archival collection of adult human cryptorchid testes to determine whether AR cellular distribution and intensity of immunostaining were functions of the severity of cellular dysgenesis. The seminiferous tubule histology of cryptorchid testes collected from adults is marked by three specific patterns. 1) Seminiferous tubules are characterized as maintaining focal areas of germinal cell differentiation (albeit incomplete) that are interspersed with 2) tubules composed of Sertoli cells only, these latter cells being principally of the adult type, although dysgenetic and immature Sertoli cells may also be detected. 3) In contrast, there is a class of tubule that is characterized as being composed exclusively of Sertoli cells that are extremely dysgenetic in appearance. The majority of adult-type Sertoli cells found in the first types of tubules exhibited either robust or moderate AR staining intensity. Peritubular cells of these tubules also expressed a similar AR staining intensity. In contrast, in the more dysgenetic and immature type Sertoli cells found in the second type of tubules, the intensity of AR staining was significantly less, if not missing altogether. Finally, in the most dysgenetic tubules, Sertoli cell AR staining was never detected. To our knowledge, this is the first report in the literature that addresses the intensity of AR immunostaining in Sertoli cells of cryptorchid testes. The results presented herein are consistent with the interpretation that the intensity of AR staining in Sertoli cells diminishes as a function of the severity to which the cells are afflicted within a cryptorchid testis and that focal absence of AR expression in Sertoli cells correlates with a lack of local spermatogenesis in the tubules.
Mutation of Three Critical Amino Acids of the N-Terminal Domain of IGF-Binding Protein-3 Essential for High Affinity IGF Binding
Authors: Buckway C. K. , Wilson E. M. , Ahlsén M. , Bang P. , Oh Y. , Rosenfeld R. G.
Published: January 2014

The N-terminal domain is conserved in all members of the IGF-binding protein superfamily. Most recently, studies have demonstrated the importance of an IGF-binding protein N-terminal hydrophobic pocket for IGF binding. To examine more critically the amino acids important for IGF binding within the full-length IGF-binding protein-3 protein while minimizing changes in the tertiary structure, we targeted residues I56, L80, and L81 within the proposed hydrophobic pocket for mutation. With a single change at these sites to the nonconserved glycine there was a notable decrease in binding. A greater reduction was seen when both L80 and L81 were substituted with glycine, and complete loss of affinity for IGF-I and IGF-II occurred when all three targeted amino acids were changed to glycine. Furthermore, the ability of the IGF-binding protein-3 mutants to inhibit IGF-I-stimulated phosphorylation of its receptor was a reflection of their affinity for IGF, with the lowest affinity mutants having the least inhibitory effect.

These studies, thus, support the hypothesis that an N-terminal hydrophobic pocket is the primary site of high affinity binding of IGF to IGF-binding protein-3. The mutants provide a tool for future studies directed at IGF-dependent and IGF-independent actions of IGF-binding protein-3.
Catecholaminergic Axons Innervate LH-Releasing Hormone Immunoreactive Neurons of the Human Diencephalon
Authors: Dudás Bertalan , Merchenthaler István
Published: January 2014

Catecholamines have been shown to modulate gonadal functions via interactions with hypothalamic LH-releasing hormone (LHRH)-synthesizing neurons. To reveal the morphological background of this phenomenon, the distribution of LHRH neurons and tyrosine hydroxylase (TH)-immunoreactive (IR), catecholaminergic structures were mapped in the human diencephalon. First, the location of LHRH and TH-IR neuronal elements was analyzed, and then the relationship between the two different systems was examined. The LHRH-IR cell bodies were mainly present in the medial preoptic and infundibular areas. The TH-IR perikarya were located in the periventricular, paraventricular, and supraoptic hypothalamic nuclei and also in the median eminence. The TH-IR fibers were numerous in septal, infundibular, periventricular, and lateral hypothalamic regions. The brown, diaminobenzidine-labeled LHRH-containing perikarya were found to receive black, silver-intensified, TH-positive axon terminals in the infundibular and medial preoptic areas. However, in the preoptic and caudal parts of the diencephalon, only a few juxtapositions were noted. The present results indicate that hormone released from diencephalic LHRH-IR neurons in humans may be influenced by the central catecholaminergic system via direct synaptic mechanisms.
Inhibition of in Situ Expression of Aromatase P450 in Leiomyoma of the Uterus by Leuprorelin Acetate
Authors: Shozu Makio , Sumitani Hiroshi , Segawa Tomoya , Yang Hei-Juan , Murakami Kouichi , Inoue Masaki
Published: January 2014

We have shown that in situ estrogen synthesized in leiomyoma of the uterus plays a possible role in the promotion of leiomyoma cell growth via an autocrine/paracrine mechanism. In the present study, we demonstrated that leuprorelin acetate, a GnRH agonist widely used for treatment of uterine leiomyoma by down-regulation of pituitary-ovarian function, suppressed the expression of aromatase P450 (an estrogen synthetase) in leiomyoma cells. Given the role of in situ estrogen in leiomyoma cell growth, the inhibition of in situ estrogen synthesis may play a role in GnRH agonist-induced rapid regression of leiomyomas. Quantitative RT-PCR revealed that in women receiving no medication uterine leiomyomas express aromatase P450 mRNA at levels 20 times higher than that in the surrounding myometrium. Leuprorelin acetate treatment (1.88 mg every 4 wk, sc injection) for 12–24 wk reduced the expression of aromatase P450 mRNA in leiomyoma tissue as well as in the myometrium, to approximately one tenth of that in the myometrium of untreated women. Suppression of aromatase P450 expression was also demonstrated by Western blot analysis and aromatase activity assay of microsomal fractions prepared from leiomyomas. On the other hand, no differences in the levels of activity and mRNA of aromatase P450 were observed between leiomyoma cells obtained from women treated with and without leuprorelin acetate injections when cells were cultured ex vivo and stimulated by various combinations of stimulants such as dexamethasone + IL-1β. The addition of various concentrations of E2 did not affect the aromatase activity of leiomyoma cells, suggesting that deprivation of circulating (ovarian) estrogen is not a cause of decreased expression of aromatase during leuprorelin acetate therapy. On the other hand, 8-d treatment with leuprorelin acetate (100 nmol/liter) reduced dexamethasone + IL-1β-induced activity and a mRNA level of aromatase by 28% and 42%, respectively. These results indicated that leuprorelin acetate inhibits the expression of aromatase P450 in leiomyoma cells, which contributes to the rapid regression of leiomyoma during leuprorelin acetate therapy.
Increases in Plasma ACTH and Cortisol after Hypertonic Saline Infusion in Patients with Central Diabetes Insipidus
Authors: Itagaki Eiji , Ozawa Sachihiko , Yamaguchi Shinya , Ushikawa Kenji , Tashiro Teruaki , Katahira Hiroshi , Takizawa Makoto , Yoshimoto Katsuhiko , Murakawa Shoichiro , Ishida Hitoshi
Published: January 2014

To clarify the mechanism for the potentiation of CRH-induced ACTH response by the infusion of hypertonic saline, we investigated changes in plasma ACTH concentration after infusion of 5% hypertonic saline in five patients with untreated central diabetes insipidus (DI). Basal levels of plasma ACTH and cortisol in the DI group were not significantly different from those in normal control subjects. The infusion of hypertonic saline produced an increase in plasma arginine vasopressin (AVP) in controls, but did not elevate ACTH. However, in patients with DI, the plasma AVP concentration did not change, but circulating ACTH increased 3.6-fold (7.7 ± 1.5 to 23.0 ± 2.7 pmol/liter; P < 0.01), and plasma cortisol also increased significantly (298 ± 99 to 538 ± 124 nmol/liter; P < 0.05). Moreover, a positive correlation was observed between plasma ACTH and osmolality (r = 0.72; P < 0.005). These results indicate that ACTH secretion in DI patients is regulated by a mechanism distinct from that in healthy subjects. It seems possible that the increase in plasma osmolality promotes ACTH secretion in DI patients through AVP and/or urocortin via the hypophyseal portal system, independent of the AVP secretion from magnocellular neurons.
Expression of Wilms’ Tumor Suppressor Gene (WT1) in Human Endometrium: Regulation through Decidual Differentiation
Authors: Makrigiannakis Antonis , Coukos George , Mantani Anastasia , Prokopakis Prokopis , Trew Geoffrey , Margara Raul , Winston Robert , White John
Published: January 2014

The Wilms’ tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract and functions as a tissue-specific developmental regulator. In addition to its gene-regulatory function through DNA binding properties, WT-1 also regulates transcription by formation of protein-protein complexes. These properties place WT-1 as a major regulator of cell growth and differentiation. In view of these observations, we studied WT1 mRNA and protein in human endometrial extracts and in endometrial stromal cells (ESCs) differentiating into decidual cells in vitro, by RT-PCR and Western blotting, respectively. WT1 protein expression was also studied in situ in the proliferative and the secretory phase of the menstrual cycle in the early pregnant state. Analysis by PCR of total RNA prepared from human ESCs demonstrated the presence of WT1 mRNA and four WT1 mRNA splice variants. Western blot analysis of nuclear protein extracts from ESCs yielded one immunoreactive protein of the expected size (approximately 52–54 kDa) recognized by the WT1 antibody. Immunohistochemical staining showed that WT1 protein is localized only to nuclei of human endometrial stromal cells. It remains constant in the proliferative and the secretory phase of the menstrual cycle and is increased remarkably during decidualization in early pregnancy. ESCs decidualized in vitro were investigated for WT-1 expression, which confirmed that decidualizing stimuli (E2, medroxy-progesterone-acetate, and relaxin for 12 d or cAMP and progesterone for 1–4 d) induced WT-1 mRNA (P < 0.05) and increased protein levels (P < 0.05). These data indicate that in humans the WT1 gene is expressed in ESCs and its mRNA and protein levels remain constant in the proliferative and the secretory phase of the menstrual cycle and that WT1 mRNA and protein expression increases significantly in ESCs when these cells differentiate into decidual cells.
The Importance of Adrenocortical Glucocorticoids for Adrenomedullary and Physiological Response to Stress: A Study in Isolated Glucocorticoid Deficiency
Authors: Zuckerman-Levin Nehama , Tiosano Dov , Eisenhofer Graeme , Bornstein Stefan , Hochberg Ze’ev
Published: January 2014

Glucocorticoids are required for the normal functioning of chromaffin cells and their capacity to produce epinephrine. This was modeled in a unique clinical syndrome of isolated glucocorticoid deficiency due to unresponsiveness to ACTH. The working hypotheses were that in patients with isolated glucocorticoid deficiency, adrenomedullary epinephrine would be suppressed despite replacement therapy; that norepinephrine might show a compensatory response; and that the physiological response to stress would reflect these changes. Toward these hypotheses, patients with ACTH unresponsiveness on glucocorticoid replacement were subjected to three levels of acute stress: assumption of upright posture, cold pressor, and exercise. Their catecholamine and physiological response were monitored. Patients with isolated glucocorticoid deficiency of this study had severe adrenomedullary dysfunction, characterized by a minimal resting production of epinephrine (6 ± 2 pg/ml compared with 64 ± 22 pg/ml of the controls) and a minimal response to stress. A slight compensatory increase of norepinephrine was found in response to cold pressor test (754 ± 200 pg/ml compared with 431 ± 73 pg/ml of the control). The physiological response is characterized by low systolic blood pressure and high pulse rate in rest and mild stress and in a pressor response to exercise (diastolic 87 ± 5 mm Hg, compared with 73 ± 2 mm Hg of the control). It is concluded that intra-adrenal glucocorticoids are essential for epinephrine secretion, that norepinephrine may be compensatory, and that these result in a distinct physiological response. The implications of the pressor response to exercise, the declining pulse pressure, and the increased pulse response insinuate a lower physical fitness in patients with adrenal insufficiency.
Assessment of Growth Hormone Dynamics in Human Immunodeficiency Virus-Related Lipodystrophy1
Authors: Rietschel Petra , Hadigan Colleen , Corcoran Colleen , Stanley Takara , Neubauer Gregory , Gertner Joseph , Grinspoon Steven
Published: January 2014

Human immunodeficiency virus (HIV) lipodystrophy (LIPO) is characterized by increased visceral adiposity, peripheral fat atrophy, dyslipidemia, and insulin resistance. GH concentrations are known to vary inversely with excess weight and body fat but have not been investigated in HIV lipodystrophy. Twenty-one subjects with HIV LIPO, 20 HIV-infected nonlipodystrophy subjects (NONLIPO), and 20 control (C) subjects were prospectively recruited for this study and compared.

Subjects in the three groups were all male, age-matched [median, 47 yr old (interquartile range, 37–50) LIPO; 41 (37–44) NONLIPO; and 43 (37–49) C], and body mass index-matched [median, 24.3 kg/m2 (interquartile range, 22.2–26.6) LIPO; 24.4 (23.3–25.9) NONLIPO; and 24.8 (22.7–26.1) C] (P> 0.05 for all comparisons). Visceral abdominal fat [16,124 mm2 (11,246–19,790) LIPO; 7,559 (5,134–11,201) NONLIPO; and 8,803 (6,165–11,623) C; P < 0.01 LIPO vs. NONLIPO and LIPO vs. C] and the ratio of visceral abdominal fat to sc abdominal fat [1.37 (0.71–2.44) LIPO vs. 0.57 (0.47–0.78) NONLIPO vs. 0.55 (0.41–0.71) C, P < 0.01 LIPO vs. NONLIPO and LIPO vs. C] were significantly increased in the LIPO subjects but were not significantly different between NONLIPO and C. The mean overnight GH concentration, determined from frequent sampling every 20 min (from 2000 h to 0800 h) was decreased in the LIPO subjects [0.38 μg/L (0.13–0.67) LIPO vs. 0.96 (0.53–1.30) NONLIPO vs. 0.81 (0.49–1.03) C, P < 0.05 LIPO vs. NONLIPO and LIPO vs. C] and not significantly different between NONLIPO and C. Pulse analysis demonstrated decreased baseline GH [0.08 μg/L (0.06–0.21) LIPO vs. 0.19 (0.10–0.32) NONLIPO vs. 0.17 (0.12–0.57) C, P < 0.05 LIPO vs. NONLIPO and LIPO vs. C] and GH peak amplitude [1.06μ g/L (0.46–1.94) LIPO vs. 2.47 (1.22–3.43) NONLIPO and 2.27 (1.36–4.25) C, P < 0.05 LIPO vs. NONLIPO and LIPO vs. C] in the LIPO subjects but no significant difference in pulse frequency. No significant differences were observed between NONLIPO and C for any GH parameter. Insulin-like growth factor-I was not different between the groups. Total body fat (r = −0.40, P = 0.01) and visceral fat (r = −0.58, P = 0.0001) correlated inversely with mean overnight GH concentrations in the HIV-infected patients. In a multivariate regression model, controlling for age, body mass index, body fat, and visceral fat, only visceral fat was a significant predictor of mean GH concentrations (P = 0.0036, r2 for model = 0.40).

These data demonstrate normal GH pulse frequency and insulin-like growth factor-I concentrations but reduced mean GH concentrations, basal GH concentrations, and GH pulse amplitude in patients with HIV lipodystrophy. Increased visceral adiposity is the strongest predictor of reduced GH concentrations in HIV lipodystrophy. Further studies are necessary to determine the clinical significance of reduced GH in patients with HIV lipodystrophy.