Comparative inhibition of the GH/IGF-I axis obtained with either the targeted secretion inhibitor SXN101959 or the somatostatin analog octreotide in growing male rats.

Abnormally high GH/IGF-I levels, most often caused by adenomas arising from pituitary somatotrophs, generate deleterious effects. We recently described a targeted secretion inhibitor (SXN101742) comprising a GHRH domain and the endopeptidase domain of botulinum toxin serotype D (GHRH-light chain endopeptidase type D domain [LC/D] associated to a heavy chain translocation domain [HN]) able to down-regulate the GH/IGF-I axis. In the present study, we compared the effect of a single iv bolus of a related molecule developed for clinical studies (SXN101959, 1 mg/kg) with a sc infusion of the somatostatin analog octreotide (SMS201-995, 10 µg/kg · h) to lower GH/IGF-I activity in growing male rats. Ten days after administration of SXN101959 or initiation of the octreotide infusion, body and pituitary weights, body length, GH peaks, and IGF-I production were reduced by both treatments but to a greater extent with SXN101959. In contrast to unaltered GH gene expression and increased GH storage in pituitaries from octreotide-treated rats, the inhibition of GH secretion was associated with a collapse of both GH mRNA and protein level in pituitaries from SXN101959-treated rats, in line with a specific decrease in hypothalamic GHRH production, not observed with octreotide. SXN101959 did not induce major apoptotic events in anterior pituitary and exhibited a reversible mode of action with full recovery of somatotroph cell functionality 30 days after treatment. Octreotide infusion permanently decreased ghrelin levels, whereas SXN101959 only transiently attenuated ghrelinemia. Both treatments limited bone mass acquisition and altered specifically tissues development. In conclusion, SXN101959 exerts a powerful and reversible inhibitory action on the somatotropic axis. Specific features of SXN101959, including long duration of action coupled to a strong inhibition of pituitary GH synthesis, represent advantages when treating overproduction of GH.

Early metabolic defects in dexamethasone-exposed and undernourished intrauterine growth restricted rats.

Poor fetal growth, also known as intrauterine growth restriction (IUGR), is a worldwide health concern. IUGR is commonly associated with both an increased risk in perinatal mortality and a higher prevalence of developing chronic metabolic diseases later in life. Obesity, type 2 diabetes or metabolic syndrome could result from noxious "metabolic programming." In order to better understand early alterations involved in metabolic programming, we modeled IUGR rat pups through either prenatal exposure to synthetic glucocorticoid (dams infused with dexamethasone 100 µg/kg/day, DEX) or prenatal undernutrition (dams feeding restricted to 30% of ad libitum intake, UN). Physiological (glucose and insulin tolerance), morphometric (automated tissue image analysis) and transcriptomic (quantitative PCR) approaches were combined during early life of these IUGR pups with a special focus on their endocrine pancreas and adipose tissue development. In the absence of catch-up growth before weaning, DEX and UN IUGR pups both presented basal hyperglycaemia, decreased glucose tolerance, and pancreatic islet atrophy. Other early metabolic defects were model-specific: DEX pups presented decreased insulin sensitivity whereas UN pups exhibited lowered glucose-induced insulin secretion and more marked alterations in gene expression of pancreatic islet and adipose tissue development regulators. In conclusion, these results show that before any catch-up growth, IUGR rats present early physiologic, morphologic and transcriptomic defects, which can be considered as initial mechanistic basis of metabolic programming.

A botulinum toxin-derived targeted secretion inhibitor downregulates the GH/IGF1 axis.

Botulinum neurotoxins (BoNTs) are zinc endopeptidases that block release of the neurotransmitter acetylcholine in neuromuscular synapses through cleavage of soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein receptor (SNARE) proteins, which promote fusion of synaptic vesicles to the plasma membrane. We designed and tested a BoNT-derived targeted secretion inhibitor (TSI) targeting pituitary somatotroph cells to suppress growth hormone (GH) secretion and treat acromegaly. This recombinant protein, called SXN101742, contains a modified GH-releasing hormone (GHRH) domain and the endopeptidase domain of botulinum toxin serotype D (GHRH-LHN/D, where HN/D indicates endopeptidase and translocation domain type D). In vitro, SXN101742 targeted the GHRH receptor and depleted a SNARE protein involved in GH exocytosis, vesicle-associated membrane protein 2 (VAMP2). In vivo, administering SXN101742 to growing rats produced a dose-dependent inhibition of GH synthesis, storage, and secretion. Consequently, hepatic IGF1 production and resultant circulating IGF1 levels were reduced. Accordingly, body weight, body length, organ weight, and bone mass acquisition were all decreased, reflecting the biological impact of SXN101742 on the GH/IGF1 axis. An inactivating 2-amino acid substitution within the zinc coordination site of the endopeptidase domain completely abolished SXN101742 inhibitory actions on GH and IGF1. Thus, genetically reengineered BoNTs can be targeted to nonneural cells to selectively inhibit hormone secretion, representing a new approach to treating hormonal excess.

Administration of growth hormone in selectively protein-deprived rats decreases BMD and bone strength.

INTRODUCTION: Isocaloric protein undernutrition is associated with decreased bone mass and decreased bone strength, together with lower IGF-I levels. It remains unclear whether administration of growth hormone (GH) corrects these alterations in bone metabolism. MATERIALS AND METHODS: Six-month-old female rats were fed isocaloric diets containing either 2.5% or 15% casein for 2 weeks. Bovine growth hormone (bGH, 0.5 or 2.5mg/kg of body weight) or vehicle was then administered as subcutaneous injections, twice daily, to rats on either diet for 4 weeks. At the proximal tibia, analysis of bone mineral density (BMD), maximal load and histomorphometry were performed. In addition, urinary deoxypyridinoline, plasma osteocalcin and IGF-I concentrations were measured. Weight was monitored weekly. RESULTS: bGH caused a dose-dependent increase in plasma IGF-I regardless of the dietary protein content. However, bGH dose-dependently decreased BMD and bone strength in rats fed the low-protein diet. There was no significant effect of bGH on BMD in rats fed the normal protein diet within this short-term treatment period, however bone formation as detected by histomorphometry was improved in this group but not the low-protein group. Osteoclast surface was increased in the low-protein bGH-treated animals only. Changes in bone turnover markers were detectable under both normal and low-protein diets. CONCLUSION: These results emphasize the major importance of dietary protein intake in the bone response to short-term GH administration, and highlight the need for further investigation into the effects of GH treatment in patients with reduced protein intake.

Perinatal exposure to bisphenol a alters early adipogenesis in the rat.

BACKGROUND: The causes of the current obesity pandemic have not been fully elucidated. Implication of environmental endocrine disruptors such as bisphenol A (BPA) on adipose tissue development has been poorly investigated. OBJECTIVES: The aim of the present study was to evaluate the effects of perinatal exposure to BPA on early adipose storage at weaning. METHODS: Pregnant Sprague-Dawley rats had access to drinking water containing 1 mg/L BPA from day 6 of gestation through the end of lactation. Pups were weaned on postnatal day (PND) 21. At that time, we investigated perigonadal adipose tissue of pups (weight, histology, gene expression). For the remaining animals, we recorded body weight and food intake for animals on either standard chow or a high-fat diet. RESULTS: Gestational exposure to BPA did not alter the sex ratio or litter size at birth. On PND1, the weight of male and female BPA-exposed pups was increased. On PND21, body weight was increased only in females, in which parametrial white adipose tissue (pWAT) weight was increased about 3-fold. This excess of pWAT was associated with adipocyte hypertrophy and overexpression of lipogenic genes such as C/EBP-alpha (CAAT enhancer binding protein alpha), PPAR-gamma (peroxisome proliferator-activated receptor gamma), SREBP-1C (sterol regulatory element binding protein-1C), LPL (lipoprotein lipase), FAS (fatty acid synthase), and SCD-1 (stearoyl-CoA desaturase 1). In addition, gene expression of SREBP-1C, FAS, and ACC (acetyl-CoA carboxylase) was also increased in liver from BPA-exposed females at PND21, without a change in circulating lipids and glucose. After weaning, perinatal BPA exposure predisposed to overweight in a sex- and diet-dependent manner. We observed no change in food intake due to perinatal BPA exposure in rats on either standard chow or a high-fat diet. CONCLUSIONS: Perinatal exposure to a low dose of BPA increased adipogenesis in females at weaning. Adult body weight may be programmed during early life, leading to changes dependent on the sex and the nutritional status. Although further studies are required to understand the mechanisms of BPA action in early life, these results are particularly important with regard to the increasing prevalence of childhood obesity and the context-dependent action of endocrine disruptors.

Prenatal nicotine exposure and the programming of metabolic and cardiovascular disorders.

Presently, a growing interest is focused on the origins of the "Metabolic Syndrome", a cluster of several metabolic disorders linking obesity, dyslipidemia, hypertension and type 2 diabetes mellitus. Clearly, genetic predisposition and deleterious lifestyle, including low physical activity and hypercaloric alimentation, have an influence on the occurrence of the Metabolic Syndrome. However, recent data suggest that the Metabolic Syndrome could also be "programmed" during intrauterine life by diverse insults to the growing foetus. Nicotine is the main stimulant and dependence-forming alkaloid found in tobacco, and despite medical advice, statistics show that 20-30% of female smokers continue the habit during gestation, representing around 10% of all pregnancies. In consequence, nicotine is one of the most universally dangerous chemicals to which developing foetuses are exposed. The present review focuses on recent epidemiological surveys and experimental animal studies that provide evidences indicating that pre- and postnatal nicotine exposure might be a contributing factor for the occurrence of metabolic disorders later in life.

Prenatal nicotine exposure alters early pancreatic islet and adipose tissue development with consequences on the control of body weight and glucose metabolism later in life.

Despite medical advice, 20-30% of female smokers continue to smoke during pregnancy. Epidemiological studies have associated maternal smoking with increased risk of obesity and type-2 diabetes in the offspring. In the present study, we investigated the impact of prenatal nicotine exposure (3 mg/kg in Sprague Dawley rats via osmotic Alzet minipumps) on the early endocrine pancreas and adipose tissue development in rat pups before weaning. Body weight, fat deposition, food intake and food efficiency, cold tolerance, spontaneous physical activity, glucose utilization, and insulin sensitivity were also examined at adulthood. Prenatal nicotine exposure led to a decrease in endocrine pancreatic islet size and number at 7 d of life (postnatal d 7), which corroborates with a decrease in gene expression of specific transcription factors such as pancreatic and duodenal homeobox 1, Pax-6, Nkx6.1, and of hormones such as insulin and glucagon. The prenatal nicotine exposure also led to an increase in epididymal white adipose tissue weight at weaning (postnatal d 21), and marked hypertrophy of adipocytes, with increased gene expression of proadipogenic transcription factors such as CAAT-enhancer-binding protein-alpha, peroxisome proliferator activated receptor-gamma, and sterol regulatory element binding protein-1C. These early tissue alterations led to significant metabolic consequences, as shown by increased body weight and fat deposition, increased food efficiency on high-fat diet, cold intolerance, reduced physical activity, and glucose intolerance combined with insulin resistance observed at adulthood. These results prove a direct association between fetal nicotine exposure and offspring metabolic syndrome with early signs of dysregulations of adipose tissue and pancreatic development.

Delayed resolution of acute inflammation during zymosan-induced arthritis in leptin-deficient mice.

The severity of antigen-induced arthritis (AIA) is decreased in leptin-deficient ob/ob mice. However, joint inflammation in AIA depends on the immune response, which is impaired in ob/ob mice. In the present study we investigated the effects of leptin deficiency on zymosan-induced arthritis (ZIA), which is independent of adaptive immunity. Arthritis was induced by injection of zymosan into the knee joint. Joint swelling was similar after 6 and 24 hours in ob/ob and control mice. However, it remained elevated in ob/ob animals on day 3 whereas values normalized in controls. Histology revealed similar articular lesions in all animals on day 3, but on days 14 and 21 arthritis tended to be more severe in ob/ob mice. The acute phase response, reflected by circulating levels of IL-6 and serum amyloid A, was also more pronounced in ob/ob mice, although corticosterone was significantly elevated in these animals. Similar results were obtained in leptin receptor-deficient db/db mice. Thus, in contrast to AIA, ZIA is not impaired in leptin-deficient animals. On the contrary, resolution of acute inflammation appears to be delayed in the absence of leptin or leptin signalling, suggesting that chronic leptin deficiency interferes with adequate control of the inflammatory response in ZIA.

Mineralo- and glucocorticoid receptor mrnas are differently regulated by corticosterone in the rat hippocampus and anterior pituitary.

In most cell lines and animal tissues, glucocorticoid receptors undergo downregulation after exposure to corticosterone. However, corticosterone treatment has not shown a consistent effect on mineralocorticoid (MR) and glucocorticoid receptors (GR) in the hippocampus, and it has been rarely assessed in the anterior pituitary. In this study we investigated dose-dependent effects of corticosterone on MR and GR mRNAs in the hippocampus and anterior pituitary. Adrenalectomized rats substituted with corticosterone in drinking fluid were injected subcutaneously with vehicle or 1, 10, 50, 100, or 200 mg of corticosterone, and sacrificed 4 h later. In the hippocampus we found a progressive decrease in MR and GR mRNAs with increasing doses of corticosterone. This was significant with 50 and 100 mg corticosterone for MR mRNA and with 10-200 mg corticosterone for GR mRNA at plasma corticosterone levels above 30 microg/dl. The anterior pituitary did not show significant changes at any dose. A time-course with 2 mg of corticosterone (non-response dose range at 4 h) revealed a significant decrease in MR and GR mRNAs in the hippocampus 8 h after the subcutaneous injection. In the anterior pituitary both mRNAs showed an increase that was significant 24 h after injection for MR and from 8 to 24 h for GR. In the hippocampus, adrenalectomy (absence of corticosterone) induced a significant increase in MR and GR mRNAs on day 3, but not on days 1, 8 and 21 after adrenalectomy. In the anterior pituitary there were no significant changes at any time after adrenalectomy. In summary, we have found an in vivo corticosterone dose- and time-dependent downregulation of MR and GR mRNAs in the hippocampus, whereas anterior pituitary MRs and GRs seem relatively insensitive to the excess or the absence of corticosterone, suggesting the lack of an autoregulatory effect in this tissue. Significant mRNA changes appearing later in time could suggest a secondary response via a glucocorticoid-induced gene product. Corticosteroid receptor downregulation in the hippocampus could prevent overstimulation or tissue damage when plasma corticosterone is high, while increased corticosteroid receptors in the anterior pituitary could buffer the excessive brain drive on the pituitary during chronic stress or pathological conditions associated with increased plasma glucocorticoids, such as depression.

Chronic neuropeptide Y infusion into the lateral ventricle induces sustained feeding and obesity in mice lacking either Npy1r or Npy5r expression.

Neuropeptide Y (NPY) is a powerful orexigenic neurotransmitter. The NPY Y1 and Y5 receptors have been implicated in mediating the appetite-stimulating activity of NPY. To further investigate the importance of these two receptors in NPY-induced hyperphagia after chronic central administration, we used mice lacking either Npy1r or Npy5r expression. NPY infusion into the lateral ventricle of wild-type mice stimulated food intake and induced obesity over a 7-d period. Fat pad weight as well as plasma insulin, leptin, and corticosterone levels were strongly increased in NPY-treated mice. In addition, NPY infusion resulted in a significant decrease in hypothalamic NPY and proopiomelanocortin expression. Interestingly, the lack of either Npy1r or Npy5r expression in knockout mice did not affect such feeding response to chronic NPY infusion. Moreover, the obesity syndrome that developed in these animals was similar to that in wild-type animals. Taken together, these data strongly suggest biological redundancies between Y1 and Y5 receptor signaling in the NPY-mediated control of food intake.

Chronic corticotropin-releasing hormone and vasopressin regulate corticosteroid receptors in rat hippocampus and anterior pituitary.

Corticotropin-releasing hormone (CRH) and vasopressin (AVP) participate in the endocrine, autonomic, immunological and behavioral response to stress. CRH and AVP receptors are found in hippocampus and anterior pituitary, where mineralocorticoid (MR) and glucocorticoid (GR) receptors are abundant. We investigated the possible influence of CRH and AVP on the regulation of MR and GR in both tissues. CRH, AVP, or their antagonists were administered to adrenalectomized rats substituted with corticosterone, to avoid interference with adrenal secretion. Repeated i.c.v. oCRH injections (10 microgram) for 5 days significantly decreased MR and GR mRNA in hippocampus and MR mRNA in anterior pituitary. AVP significantly increased both corticosteroid receptor mRNAs, as repeated i.c.v. injections (5 microgram) for 5 days in hippocampus, and as continuous i.c.v. infusion (10 ng/h/5 days) in anterior pituitary. The i.c.v. infusion of 5 or 10 microgram/day of the alpha-helical CRH antagonist during intermittent restraint stress (5 days), induced a significant decrease in hippocampal MR binding. In anterior pituitary, 5 microgram/day significantly decreased MR binding, while 10 microgram/day significantly increased GR binding. Under the same conditions of stress, the infusion of 15 microgram/day of the vasopressin V1a/1b receptor antagonist [dP Tyr (Me)(2)AVP] significantly increased MR and GR binding in hippocampus and anterior pituitary; 5 microgram/day significantly decreased pituitary MR binding. Our results show that CRH and AVP regulate MR and GR in hippocampus and anterior pituitary. This reveals another important function of CRH and AVP, which could be relevant to understand stress adaptation and the pathophysiology of stress-related disorders like major depression.

Chronic inhibition of circulating dipeptidyl peptidase IV by FE 999011 delays the occurrence of diabetes in male zucker diabetic fatty rats.

Acute suppression of dipeptidyl peptidase IV (DPP-IV) activity improves glucose tolerance in the Zucker fatty rat, a rodent model of impaired glucose tolerance, through stabilization of glucagon-like peptide (GLP)-1. This study describes the effects of a new and potent DPP-IV inhibitor, FE 999011, which is able to suppress plasma DPP-IV activity for 12 h after a single oral administration. In the Zucker fatty rat, FE 999011 dose-dependently attenuated glucose excursion during an oral glucose tolerance test and increased GLP-1 (7-36) release in response to intraduodenal glucose. Chronic treatment with FE 999011 (10 mg/kg, twice a day for 7 days) improved glucose tolerance, as suggested by a decrease in the insulin-to-glucose ratio. In the Zucker diabetic fatty (ZDF) rat, a rodent model of type 2 diabetes, chronic treatment with FE 999011 (10 mg/kg per os, once or twice a day) postponed the development of diabetes, with the twice-a-day treatment delaying the onset of hyperglycemia by 21 days. In addition, treatment with FE 999011 stabilized food and water intake to prediabetic levels and reduced hypertriglyceridemia while preventing the rise in circulating free fatty acids. At the end of treatment, basal plasma GLP-1 levels were increased, and pancreatic gene expression for GLP-1 receptor was significantly upregulated. This study demonstrates that DPP-IV inhibitors such as FE 999011 could be of clinical value to delay the progression from impaired glucose tolerance to type 2 diabetes.

Pharmacological profile of a new, potent, and long-acting gonadotropin-releasing hormone antagonist: degarelix.

We describe the pharmacological profile in rats and monkeys of degarelix (FE200486), a member of a new class of long-acting gonadotropin-releasing hormone (GnRH) antagonists. At single subcutaneous injections of 0.3 to 10 microg/kg in rats, degarelix produced a dose-dependent suppression of the pituitary-gonadal axis as revealed by the decrease in plasma luteinizing hormone (LH) and testosterone levels. Duration of LH suppression increased with the dose: in the rat, significant suppression of LH lasted 1, 2, and 7 days after a single subcutaneous injection of degarelix at 12.5, 50, or 200 microg/kg, respectively. Degarelix fully suppressed plasma LH and testosterone levels in the castrated and intact rats as well as in the ovariectomized rhesus monkey for more than 40 days after a single 2-mg/kg subcutaneous injection. In comparative experiments, degarelix showed a longer duration of action than the recently developed GnRH antagonists abarelix, ganirelix, cetrorelix, and azaline B. The in vivo mechanism of action of degarelix was consistent with competitive antagonism, and the prolonged action of degarelix was paralleled by continued presence of radioimmunoassayable degarelix in the general circulation. In contrast to cetrorelix and similarly to ganirelix and abarelix, degarelix had only weak histamine-releasing properties in vitro. These results demonstrate that the unique and favorable pharmacological properties of degarelix make it an ideal candidate for the management of sex steroid-dependent pathologies requiring long-term inhibition of the gonadotropic axis.

Synthesis, purification and structural and functional characterization of recombinant form of a common genetic variant of human luteinizing hormone.

A common genetic variant (V) of luteinizing hormone (LH), with two mutations (Trp(8)Arg and Ile(15)Thr) and an extra glycosylation consensus site (Asn(13)-Ala-Thr), is associated with abnormalities of reproductive function. To address the molecular basis of the functional differences between V- and wild-type (WT)-LH, recombinant (rec) forms of WT- and V-LH were synthesized in human embryonic kidney (HEK 293) cells. The rec hormones synthesized were rigorously purified employing affinity, immunoaffinity and ion exchange chromatographies (final purity approximately 12 000 IU/mg, 180-fold purification, 28% recovery). Functional properties of the hormone preparations were compared in vitro and in vivo. The molecular size of both rec LHs was 31 kDa, as determined by SDS-PAGE. Although the mutations in V-LHbeta did not significantly affect the affinity of LH receptor (LHR) binding (Kd approximately 0.4 nmol/L), V-LH had higher in vitro biopotency than WT-LH, in terms of mLTC-1 mouse Leydig tumor cell cAMP and progesterone (P) production, and steroidogenic acute regulatory protein (StAR) expression. In addition, in HEK 293 cells expressing the human LHR, V-LH demonstrated 1.8-fold higher response of inositol trisphosphate (IP(3)) production than WT-LH. Furthermore, HEK 293 cells expressing the ElK1 trans-reporting plasmids displayed 2.7-fold greater luciferase response to V-LH than WT-LH, documenting stimulation of the mitogen-activated protein kinase (MAPK) pathway. The in vivo half-life of V-LH was clearly faster (5-9 min) than that of WT-LH (12-22 min) and human chorionic gonadotropin (hCG; 50-70 min), when injected into rat circulation. It is worth noting that analysis by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) demonstrated clear differences in structures of carbohydrate side chains attached to the two forms of rec LHs, including incomplete processing of high mannose glycans (Man(5,8,9)) in V-LH, suggesting different pathways in its intracellular trafficking. Collectively, the present findings provide the molecular basis for the qualitative and quantitative differences in LH action that are observed in carriers of the V-LHbeta allele.

Cardiac and renal effects of growth hormone in volume overload-induced heart failure: role of NO.

Growth hormone (GH) application is a new strategy in the treatment of heart failure. However, clinical and experimental investigations have shown contradictory effects of GH on cardiac performance. We tested the hypothesis that GH could improve cardiac and renal function in volume overload-induced heart failure. The effect of 4 weeks of GH treatment (2 mg/kg daily) was investigated in Wistar rats with aortocaval shunt. GH application did not influence left ventricular contractility and end-diastolic pressure in rats with aortocaval shunt. In contrast, GH treatment normalized impaired diuresis (vehicle 10.8+/-0.6 mL/d, GH 15.8+/-0.7 mL/d; P<0.05) and sodium excretion (vehicle 1.5+/-0.1 mmol/d, GH 2.2+/-0.1 mmol/d; P<0.001) in shunt-operated rats, with a similar increase of fractional sodium excretion. The urinary excretion of cGMP, the second messenger of atrial natriuretic peptide and NO, was higher in animals with shunts than in sham-operated animals and was further increased by GH (vehicle 293+/-38 nmol/d, GH 463+/-57 nmol/d; P<0.01). Although the atrial natriuretic peptide plasma levels were unchanged after GH, the excretion of NO metabolites (nitrate/nitrite) was elevated (vehicle 2020+/-264 nmol/d, GH 2993+/-375 nmol/d; P<0.05) in parallel with increased renal mRNA levels of inducible NO synthase 2. The changes of renal function after GH and the increased excretion of NO metabolites and cGMP were abolished by simultaneous treatment with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. GH treatment did not influence cardiac function in rats with aortocaval shunts. However, GH improved renal function by increasing diuresis and sodium excretion. The responsible mechanism might be the enhanced activity of the renal NO system.