Growth hormone replacement therapy regulates microRNA-29a and targets involved in insulin resistance

Replacement of growth hormone (GH) in patients suffering from GH deficiency (GHD) offers clinical benefits on body composition, exercise capacity, and skeletal integrity. However, GH replacement therapy (GHRT) is also associated with insulin resistance, but the mechanisms are incompletely understood. We demonstrate that in GH-deficient mice (growth hormone-releasing hormone receptor (Ghrhr)(lit/lit)), insulin resistance after GHRT involves the upregulation of the extracellular matrix (ECM) and the downregulation of microRNA miR-29a in skeletal muscle. Based on RNA deep sequencing of skeletal muscle from GH-treated Ghrhr(lit/lit) mice, we identified several upregulated genes as predicted miR-29a targets that are negative regulators of insulin signaling or profibrotic/proinflammatory components of the ECM. Using gain- and loss-of-function studies, five of these genes were confirmed as endogenous targets of miR-29a in human myotubes (PTEN, COL3A1, FSTL1, SERPINH1, SPARC). In addition, in human myotubes, IGF1, but not GH, downregulated miR-29a expression and upregulated COL3A1. These results were confirmed in a group of GH-deficient patients after 4 months of GHRT. Serum IGF1 increased, skeletal muscle miR-29a decreased, and miR-29a targets were upregulated in patients with a reduced insulin response (homeostatic model assessment of insulin resistance (HOMA-IR)) after GHRT. We conclude that miR-29a could contribute to the metabolic response of muscle tissue to GHRT by regulating ECM components and PTEN. miR-29a and its targets might be valuable biomarkers for muscle metabolism following GH replacement. KEY MESSAGES GHRT most significantly affects the ECM cluster in skeletal muscle from mice. GHRT downregulates miR-29a and upregulates miR-29a targets in skeletal muscle from mice. PTEN, COL3A1, FSTL1, SERPINH1, and SPARC are endogenous miR-29a targets in human myotubes. IGF1 decreases miR-29a levels in human myotubes. miR-29a and its targets are regulated during GHRT in skeletal muscle from humans.

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in Cpefat mice associated with a carboxypeptidase E mutation

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia–diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.

Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion

The classic view for hypothalamic regulation of anterior pituitary (AP) hormone secretion holds that release of each AP hormone is controlled specifically by a corresponding hypothalamic-releasing hormone (HRH). In this scenario, binding of a given HRH (thyrotropin-, growth hormone-, corticotropin-, and luteinizing hormone-releasing hormones) to specific receptors in its target cell increases the concentration of cytosolic Ca2+ ([Ca2+]i), thereby selectively stimulating the release of the appropriate hormone. However, “paradoxical” responses of AP cells to the four well-established HRHs have been observed repeatedly with both in vivo and in vitro systems, raising the possibility of functional overlap between the different AP cell types. To explore this possibility, we evaluated the effects of HRHs on [Ca2+]i in single AP cells identified immunocytochemically by the hormone they stored. We found that each of the five major AP cell types contained discrete subpopulations that were able to respond to several HRHs. The relative abundance of these multi-responsive cells was 59% for lactotropes, 33% for thyrotropes, and in the range of 47–55% for gonadotropes, corticotropes, and somatotropes. Analysis of prolactin release from single living cells revealed that each of the four HRHs tested were able to induce hormone release from a discrete lactotrope subpopulation, the size of which corresponded closely to that in which [Ca2+]i changes were induced by the same secretagogues. When viewed as a whole, our diverse functional measurements of multi-responsiveness suggest that hypothalamic control of pituitary function is more complicated than previously envisioned. Moreover, they provide a cellular basis for the so-called “paradoxical” behavior of pituitary cells to hypothalamic hypophysiotropic agents.

Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.

Design and biological activities of L-163,191 (MK-0677): a potent, orally active growth hormone secretagogue.

A potent, orally active growth hormone (GH) secretagogue L-163,191 belonging to a recently synthesized structural class has been characterized. L-163,191 releases GH from rat pituitary cells in culture with EC50 = 1.3 +/- 0.09 nM and is mechanistically indistinguishable from the GH-releasing peptide GHRP-6 and the prototypical nonpeptide GH secretagogue L-692,429 but clearly distinguishable from the natural GH secretagogue, GH-releasing hormone. L-163,191 elevates GH in dogs after oral doses as low as 0.125 mg/kg and was shown to be specific in its release of GH without significant effect on plasma levels of aldosterone, luteinizing hormone, thyroxine, and prolactin after oral administration of 1 mg/kg. Only modest increases in cortisol were observed. Based on these properties, L-163,191 has been selected for clinical studies.

Ovarian suppression using luteinizing hormonereleasing hormone agonists during chemotherapy to preserve ovarian function and fertility of breast cancer patients: A meta-analysis of randomized studies

Background: The role of temporary ovarian suppression with luteinizing hormone-releasing hormone agonists (LHRHa) in the prevention of chemotherapy-induced premature ovarian failure (POF) is still controversial. Our meta-analysis of randomized, controlled trials (RCTs) investigates whether the use of LHRHa during chemotherapy in premenopausal breast cancer patients reduces treatment-related POF rate, increases pregnancy rate, and impacts disease-free survival (DFS). Methods: A literature search using PubMed, Embase, and the Cochrane Library, and the proceedings of major conferences, was conducted up to 30 April 2015. Odds ratios (ORs) and 95% confidence intervals (CIs) for POF (i.e. POF by study definition, and POF defined as amenorrhea 1 year after chemotherapy completion) and for patients with pregnancy, as well hazard ratios (HRs) and 95% CI for DFS, were calculated for each trial. Pooled analysis was carried out using the fixed- and random-effects models. Results: A total of 12 RCTs were eligible including 1231 breast cancer patients. The use of LHRHa was associated with a significant reduced risk of POF (OR 0.36, 95% CI 0.23-0.57; P < 0.001), yet with significant heterogeneity (I2 = 47.1%, Pheterogeneity = 0.026). In eight studies reporting amenorrhea rates 1 year after chemotherapy completion, the addition of LHRHa reduced the risk of POF (OR 0.55, 95% CI 0.41-0.73, P < 0.001) without heterogeneity (I2 = 0.0%, Pheterogeneity = 0.936). In five studies reporting pregnancies, more patients treated with LHRHa achieved pregnancy (33 versus 19 women; OR 1.83, 95% CI 1.02-3.28, P = 0.041; I2 = 0.0%, Pheterogeneity = 0.629). In three studies reporting DFS, no difference was observed (HR 1.00, 95% CI 0.49-2.04, P = 0.939; I2 = 68.0%, Pheterogeneity = 0.044). Conclusion: Temporary ovarian suppression with LHRHa in young breast cancer patients is associated with a reduced risk of chemotherapy-induced POF and seems to increase the pregnancy rate, without an apparent negative consequence on prognosis.

Postoperative Treatment in a Patient After Hemithyroidectomy: the Therapeutic Challenges of a Hidden Thyrotropinoma

Objectives: We report the unusual case of a patient with a thyrotropinoma, discovered after a hemithyroidectomy for a suspicious thyroid nodule, and its therapeutic challenges. Materials and methods: In a patient who underwent hemithyroidectomy for cold thyroid nodule, hyperthyroid symptoms persisted, despite stopping levothyroxine treatment. Further investigation was carried out through the following laboratory tests: thyroid-stimulating hormone (TSH) test; free thyroxine (fT4) test; and the thyrotropin releasing hormone (TRH) test. A pituitary magnetic resonance imaging (MRI) scan and genetic analysis was also carried out. The test results confirmed the diagnosis of a thyrotropinoma. Results: Treatment with long-acting somatostatin analogues normalised thyroid hormones and symptoms of hyperthyroidism. Conclusion: The diagnostic approach to the thyroid nodule should include a detailed clinical and biochemical examination. Initial biochemical evaluation by TSH alone does not allow detecting inappropriate TSH secretion that may increase the risk of thyroid malignancy. In case of a thyrotropinoma, the ideal treatment consists of combined care of central and peripheral thyroid disease.

Intermittent Fasting Induces Hypothalamic Modifications Resulting In Low Feeding Efficiency, Low Body Mass And Overeating.

Intermittent fasting (IF) is an often-used intervention to decrease body mass. In male Sprague-Dawley rats, 24 hour cycles of IF result in light caloric restriction, reduced body mass gain, and significant decreases in the efficiency of energy conversion. Here, we study the metabolic effects of IF in order to uncover mechanisms involved in this lower energy conversion efficiency. After 3 weeks, IF animals displayed overeating during fed periods and lower body mass, accompanied by alterations in energy-related tissue mass. The lower efficiency of energy use was not due to uncoupling of muscle mitochondria. Enhanced lipid oxidation was observed during fasting days, whereas fed days were accompanied by higher metabolic rates. Furthermore, an increased expression of orexigenic neurotransmitters AGRP and NPY in the hypothalamus of IF animals was found, even on feeding days, which could explain the overeating pattern. Together, these effects provide a mechanistic explanation for the lower efficiency of energy conversion observed. Overall, we find that IF promotes changes in hypothalamic function that explain differences in body mass and caloric intake.

Correlation Between Testosterone And Psa Kinetics In Metastatic Prostate Cancer Patients Treated With Diverse Chemical Castrations.

To assess total testosterone and prostatic-specific antigen (PSA) kinetics among diverse chemical castrations, advanced-stage prostate cancer patients were randomized into three groups of 20: Group 1, Leuprolide 3.75 mg; Group 2, Leuprolide 7.5 mg; and Group 3, Goserelin 3.6 mg. All groups were treated with monthly application of the respective drugs. The patients' levels of serum total testosterone and PSA were evaluated at two time periods: before the treatment and 3 months after the treatment. Spearman's rank correlation coefficient was utilized to verify the hypothesis of linear correlation between total testosterone and PSA levels. At the beginning the patients' age, stage, grade, PSA, and total testosterone were similar within the three groups, with median age 72, 70, and 70 years in Groups 1, 2, and 3, respectively. Three months after the treatment, patients who received Leuprolide 7.5 mg presented significantly lower median total testosterone levels compared with Goserelin 3.6 mg and Leuprolide 3.75 mg (9.5 ng/dL vs. 20.0 ng/dL vs. 30.0 ng/dL, respectively; p = .0072), while those who received Goserelin 3.6 mg presented significantly lower PSA levels compared with Leuprolide 7.5 mg and Leuprolide 3.75 mg (0.67 vs. 1.86 vs. 2.57, respectively; p = .0067). There was no linear correlation between total testosterone and PSA levels. Overall, regarding castration levels of total testosterone, 28.77% of patients did not obtain levels ≤50 ng/dL and 47.80% did not obtain levels ≤20 ng/dL. There was no correlation between total testosterone and PSA kinetics and no equivalence among different pharmacological castrations.

Hypothalamic-pituitary axis and peripheral tissue responses to TRH stimulation and Liothyronine suppression tests in normal subjects evaluated by current methods

Objective: To reevaluate the responses of thyrotropin-releasing hormone ( TRH) stimulation test in baseline condition as well as after the administration of graded supraphysiological doses of liothyronine ( L- T-3) in normal subjects. Design: To assess various parameters related to the hypothalamic-pituitary axis and peripheral tissue responses to L- T-3 in 22 normal individuals ( median age: 30.5 years). Subjects were submitted to an intravenous TRH test at baseline condition and also to the oral administration of sequential and graded doses of L- T-3 ( 50, 100, and 200 mu g/day), each given over 3 days, at an outpatient clinic. Blood samples were obtained for thyrotropin (TSH) and prolactin (PRL) at basal and then 15, 30, and 60 minutes after the TRH injection. Effects of L- T3 administration on cholesterol, creatine kinase, retinol, ferritin, and sex hormone-binding globulin ( SHBG) were also measured at basal and after the oral administration of L- T-3. Main outcome: TRH administration resulted in an increase of 4-to 14-fold rise in serum TSH ( 8.3 +/- 2.5-fold), and in a slight rise in serum PRL concentrations ( 3.8 +/- 1.5-fold). Administration of graded doses of triiodothyronine ( T-3) resulted in a dose-dependent suppression of TSH and PRL. Basal thyroxine- binding globulin (TBG) and cholesterol levels decreased, and ferritin and SHBG increased after L- T-3 administration, while creatine kinase and retinol did not change throughout the study. There was a positive correlation between basal TSH and TSH peak response to TRH at basal condition and after each sequential L- T-3 doses. On the other hand, TSH peak response to the TRH test did not predict cholesterol, TBG, ferritin, or SHBG values. Conclusion: Using the current methods on hormone and biochemical analysis, we standardized the response of many parameters to TRH stimulation test after sequential and graded T-3 suppression test in normal subjects. Our data suggest that the evaluation of the responses of the hypothalamus-pituitary axis to TRH test as well as the impact of L- T-3 on peripheral tissues were not modified by the current methods.

Peptidase activities in the semen from the ductus deferens and uterus of the neotropical rattlesnake Crotalus durissus terrificus

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, intra- and inter-seasonal activity levels of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl-IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases as well as prolyl endopeptidase (POP) were evaluated in soluble (SF) and/or membrane-bound (MF) fractions of semen collected from the ductus deferens of the male reproductive tract and from the posterior portion of the uterus. Seminal APB, PIP and POP were detected in SF, while other peptidases were detected in SF and MF. Only the convoluted posterior uterus in winter and autumn had semen. Relative to other examined peptidases, in general, APN-PI, APN-PS and APB activities were predominant in the semen from the uterus and throughout the year in the semen from the ductus deferens, suggesting their great relevance in the seminal physiology of C. d. terrificus. The levels of peptidase activities in the ductus deferens semen varied seasonally and were different from those of semen in the uterus, suggesting that their modulatory actions on susceptible peptides are integrated to the male reproductive cycle events and spermatozoa viability of this snake.

Seasonal variation of peptidase activities in the reproductive tract of Crotalus durissus terrificus

Seasonal quantitative patterns of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolylimino (PIP) aminopeptidases and prolyl oligopeptidase (POP) activities in soluble (SF) and solubilized membrane-bound (MF) fractions from ductus deferens, vagina and uterus were studied to evaluate their relationships with the reproductive cycle and the extensive long-term spermatozoa storage (LTSS) of the Neotropical rattlesnake Crotalus durissus terrificus. APB, PIP and POP were detected only in SF, while other peptidases were detected in SF and MF. APB, APN-PI and APN-PS were predominant in most tissues in all seasons. Peptidase activities had a common pattern of increment during the dry season (winter/autumn), which coincides with the mating period (autumn) and LTSS in the female (winter), as well as the reduction of spermatozoa motility and maintenance of fertilization capacity of spermatozoa. The high CAP activity in the soluble fraction of the vagina during winter, compared to summer (time of parturition) and spring, coincides with the relaxation of this tissue. In the soluble fraction, the low PAP-1 activity of the ductus deferens coincided with its high activity in the vagina during the winter; and the inverse occurred in summer, which is consistent with the physiological process of preserving spermatozoon viability. In conclusion, the studied peptidase activities had seasonal and tissue-specific characteristics, which suggest a relevant role in the reproductive physiology of C. d. terrificus. (C) 2008 Elsevier Inc. All rights reserved.

Peptidase activities in Crotalus durissus terrificus semen

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, activity levels of representative enzymes in semen and their sensitivities to inhibitors, cofactors, and peptide hormones were evaluated. The existence of seminal fractions and the association of peptidases with these fractions were also characterized for the first time in snakes. The prominent inhibitors of aminopeptidases (APs) were amastatin for acid, basic, and neutral; bestatin for basic; and diprotin A for dipeptidyl-IV. Cystyl and prolylimino AN were similarly susceptible to the majority of these inhibitors. The basic and neutral were characterized as metallo-peptidases, acid AP was activated by MnCl(2), and cystyl, prolyl-imino, and type I pyroglutamyl were characterized as sulphydryl-dependent APs. Angiotensin II, vasotocin, bradykinin, fertilization-promoting peptide, and TRH altered the majority of these peptidase activities; these peptides are possible substrates and/or modulators of these peptidases. Peptidase activities were found in all seminal fractions: seminal plasma (SP), prostasome-like (PR) structures, and soluble (S-) and membrane-bound fractions (MFs) of spermatozoa. The levels of activity of each peptidase varied among different seminal fractions. In SP, the higher activities were puromycin-insensitive neutral and basic APs. in PR, the higher activity was puromycin-insensitive neutral AP. In spermatozoa, the higher activity in subcellular SF was puromycin-sensitive neutral, while in MF both puromycin-sensitive and -insensitive neutral AN were equally higher than the other examined peptidases. Data suggested that these peptidases, mainly basic and neutral, have a high relevance in regulating seminal functions of C. d. terrificus.

Genic expression of the uterine leiomyoma in reproductive-aged women after treatment with goserelin

Objective: To identify the genes presenting different expression in uterine leiomyomas after goserelin treatment. Design: Retrospective analyses of tissue obtained in a prospective clinical study. Setting: School of Medicine of the University of Sao Paulo. Patient(s): 30 nulliparous black women aged 20 to 45 years with symptoms of uterine leiomyoma, uterine volume over 300 mL, and surgical indications for myomectomy. Intervention(s): Fifteen patients were given a monthly dose of 3.6 mg of goserelin over 3 months before surgery (group A), and 15 patients underwent surgery without any previous treatment (group B). Five random samples from each group were analyzed using the microarray technique with the Affymetrix platform (GeneChip Rat Genome 230 2.0 Array). Main Outcome Measure(s): Quantification of transcript expression levels of uterine fibroids in patients treated or not treated with goserelin. Result(s): Of the total of 47,000 sequences that were analyzed, representing approximately 38,500 human genes already characterized, we found a differential expression of 174 genes. Of these, 70 were up-regulated (33 genes with known function) and 104 were down-regulated (65 genes with known function) in samples from group A (treated) when compared with group B (nontreated). Conclusion(s): The genic expression of uterine leiomyomas changes in women who have had goserelin treatment when compared with nontreated patients. (Fertil Steril (R) 2010; 94: 1072-7. (C) 2010 by American Society for Reproductive Medicine.)

Final height of adults with childhood-onset steroid-responsive idiopathic nephrotic syndrome

The aim of this study was to evaluate the final stature of adults with childhood-onset steroid-responsive idiopathic nephrotic syndrome (INS) and the influence of disease-related issues on the achievement of their target heights. We analyzed 60 (41 male) patients and/or their records, with a minimum age of 19 years or at a Tanner`s pubertal stage 4 for boys or status postmenarche for girls, and normal glomerular filtration rate. Mean age at first and last consultation was 5.3 +/- 2.4 years and 20.5 +/- 3.1 years, respectively. Mean follow-up period was 15.10 years. Mean cumulative dose of prednisone was 1254 +/- 831.40 mg/kg. Mean initial and final height Z scores (HtZ) were, respectively, -0.60 +/- 1.0 and -0.64 +/- 0.92 (p = 0.72). The final HtZ showed a significant correlation only with the initial HtZ and the target HtZ (THZ). Six patients achieved a final HtZ below -2, which in male patients correlated strongly to the initial HtZ and THZ. A strong correlation was demonstrated between final HtZ, initial HtZ, and THZ. INS-related issues did not prevent the final stature to reach the predicted target height.