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Context The risk of fragility fractures is increased in both type 1 and type 2 diabetes. Numerous biochemical markers reflecting bone and/or glucose metabolism have been evaluated in this context. Objective This review summarizes current data on biochemical markers in relation to bone fragility and fracture risk in diabetes. Methods A group of experts from the International Osteoporosis Foundation and European Calcified Tissue Society reviewed the literature focusing on biochemical markers, diabetes, diabetes treatments, and bone in adults. Results Although bone resorption and bone formation markers are low and poorly predictive of fracture risk in diabetes, osteoporosis drugs seem to change bone turnover markers (BTMs) in diabetics similarly to nondiabetics, with similar reductions in fracture risk. Several other biochemical markers related to bone and glucose metabolism have been correlated with bone mineral density and/or fracture risk in diabetes, including osteocyte-related markers such as sclerostin, glycated hemoglobin A1c (HbA1c) and advanced glycation end products, inflammatory markers, and adipokines, as well as insulin-like growth factor-1 and calciotropic hormones. Conclusion Several biochemical markers and hormonal levels related to bone and/or glucose metabolism have been associated with skeletal parameters in diabetes. Currently, only HbA1c levels seem to provide a reliable estimate of fracture risk, while BTMs could be used to monitor the effects of antiosteoporosis therapy.

As they age mice deficient for the ß2 adrenergic receptor (Adrb2( / ) ) maintain greater trabecular bone microarchitecture as a result of lower bone resorption and increased bone formation. The role of ß1 adrenergic receptor signaling and its interaction with ß2 adrenergic receptor on bone mass regulation however remains poorly understood. We first investigated the skeletal response to mechanical stimulation in mice deficient for ß1 adrenergic receptors and/or ß2 adrenergic receptors. Upon axial compression loading of the tibia bone density cancellous and cortical microarchitecture as well as histomorphometric bone forming indices were increased in both Adrb2( / ) and wild type (WT) mice but not in Adrb1( / ) nor in Adrb1b2( / ) mice. Moreover in the unstimulated femur and vertebra bone mass and microarchitecture were increased in Adrb2( / ) mice whereas in Adrb1( / ) and Adrb1b2( / ) double knockout mice femur bone mineral density (BMD) cancellous bone volume/total volume (BV/TV) cortical size and cortical thickness were lower compared to WT. Bone histomorphometry and biochemical markers showed markedly decreased bone formation in Adrb1b2( / ) mice during growth which paralleled a significant decline in circulating insulin like growth factor 1 (IGF 1) and IGF binding protein 3 (IGF BP3). Finally administration of the ß adrenergic agonist isoproterenol increased bone resorption and receptor activator of NF ?B ligand (RANKL) and decreased bone mass and microarchitecture in WT but not in Adrb1b2( / ) mice. Altogether these results demonstrate that ß1 and ß2 adrenergic signaling exert opposite effects on bone with ß1 exerting a predominant anabolic stimulus in response to mechanical stimulation and during growth whereas ß2 adrenergic receptor signaling mainly regulates bone resorption during aging. © 2012 American Society for Bone and Mineral Research.

OBJECTIVE:NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS) producing NADPH oxidase that regulates redox homeostasis in diverse insulin sensitive cell types. In particular NOX4 derived ROS is a key modulator of adipocyte differentiation and mediates insulin receptor signaling in mature adipocytes in vitro. Our study was aimed at investigating the role of NOX4 in adipose tissue differentiation whole body metabolic homeostasis and insulin sensitivity in vivo.DESIGN:Mice with genetic ablation of NOX4 (NOX4 deficient mice) were subjected to chow or high fat containing diet for 12 weeks. Body weight gain adiposity insulin sensitivity and adipose tissue and liver gene and protein expression were analyzed and compared with similarly treated wild type mice.RESULTS:Here we report that NOX4 deficient mice display latent adipose tissue accumulation and are susceptible to diet induced obesity and early onset insulin resistance. Obesity results from accelerated adipocyte differentiation and hypertrophy and an increase in whole body energy efficiency. Insulin resistance is associated with increased adipose tissue hypoxia inflammation and adipocyte apoptosis. In the liver more severe diet induced steatosis was observed due to the lack of proper upregulation of mitochondrial fatty acid ß oxidation.CONCLUSION:These findings identify NOX4 as a regulator of metabolic homeostasis. Moreover they indicate an anti adipogenic role for NOX4 in vivo and reveal its function as a protector against the development of diet induced obesity insulin resistance and hepatosteatosis.International Journal of Obesity advance online publication 20 March 2012; doi:10.1038/ijo.2011.279.

Patients affected by Duchenne muscular dystrophy (DMD) and dystrophic MDX mice were investigated in this study for their bone phenotype and systemic regulators of bone turnover. Micro–computed tomographic (µCT) and histomorphometric analyses showed reduced bone mass and higher osteoclast and bone resorption parameters in MDX mice compared with wild-type mice, whereas osteoblast parameters and mineral apposition rate were lower. In a panel of circulating pro-osteoclastogenic cytokines evaluated in the MDX sera, interleukin 6 (IL-6) was increased compared with wild-type mice. Likewise, DMD patients showed low bone mineral density (BMD) Z-scores and high bone-resorption marker and serum IL-6. Human primary osteoblasts from healthy donors incubated with 10% sera from DMD patients showed decreased nodule mineralization. Many osteogenic genes were downregulated in these cultures, including osterix and osteocalcin, by a mechanism blunted by an IL-6-neutralizing antibody. In contrast, the mRNAs of osteoclastogenic cytokines IL6, IL11, inhibin-βA, and TGFβ2 were increased, although only IL-6 was found to be high in the circulation. Consistently, enhancement of osteoclastogenesis was noted in cultures of circulating mononuclear precursors from DMD patients or from healthy donors cultured in the presence of DMD sera or IL-6. Circulating IL-6 also played a dominant role in osteoclast formation because ex vivo wild-type calvarial bones cultured with 10% sera of MDX mice showed increase osteoclast and bone-resorption parameters that were dampen by treatment with an IL-6 antibody. These results point to IL-6 as an important mediator of bone loss in DMD and suggest that targeted anti-IL-6 therapy may have a positive impact on the bone phenotype in these patients. © 2011 American Society for Bone and Mineral Research

Objective The aim of this study was to determine the skeletal effect of quercetin-6-C-β-D-glucopyranoside (QCG) isolated from the extract of Ulmus wallichiana and compare this effect with quercetin (Q) in a rat model of postmenopausal bone loss. Methods Murine bone marrow cells were used to study the effect of QCG or Q on osteoclast differentiation. QCG or Q (1.0 and 5.0 mg kg(-1) d(-1) doses) was administered orally to ovarietomized (OVx) rats for 12 weeks. Sham-operated + vehicle and OVx + vehicle groups served as positive and negative controls, respectively. Bone mineral density, bone microarchitecture, biomechanical strength, bone turnover markers, and uterotrophic effect were studied. One-way analysis of variance was used to test significance of effects. Results QCG at 1.0 nM significantly inhibited differentiation of multinucleated osteoclasts and expression of osteoclastogenic genes from bone marrow cells, whereas Q at 10.0 μM had comparable results. OVx rats treated with QCG exhibited significantly higher bone mass and better microarchitecture in trabecular and cortical bones compared with OVx + vehicle. QCG treatment of OVx rats had better functional impact than did Q-treated OVx rats, evident from increased bone biomechanical strength. Serum osteocalcin and urinary fragments of type 1 collagen were significantly lower in QCG-treated OVx rats compared with OVx + vehicle group. The protective effect of QCG under ovariectomy-induced bone loss setting was found to be significantly better than Q. Uterine histomorphometry parameters of OVx rats did not change with QCG treatment. Conclusions QCG improves bone biomechanical quality more effectively than Q through positive modifications of bone mineral density and bone microarchitecture without a hyperplastic effect on the uterus.

PTH stimulates osteoblastic cells to form new bone and to produce osteoblast-osteoclast coupling factors such as RANKL. Whether osteoclasts or their activity are needed for PTH anabolism remains uncertain. We treated ovariectomized huRANKL knock-in mice with a human RANKL inhibitor denosumab (DMAb), alendronate (Aln), or vehicle for 4 weeks, followed by co-treatment with intermittent PTH for 4 weeks. Loss of bone mass and microarchitecture was prevented by Aln and further significantly improved by DMAb. PTH improved bone mass, microstructure, and strength, and was additive to Aln but not to DMAb. Aln inhibited biochemical and histomorphometrical indices of bone turnover, -i.e. osteocalcin and bone formation rate (BFR) on cancellous bone surfaces-, and Dmab inhibited them further. However Aln increased whereas Dmab suppressed osteoclast number and surfaces. PTH significantly increased osteocalcin and bone formation indices, in the absence or presence of either antiresorptive, although BFR remained lower in presence of Dmab. To further evaluate PTH effects in the complete absence of osteoclasts, high dose PTH was administered to RANK−/− mice. PTH increased osteocalcin similarly in RANK−/− and WT mice. It also increased BMD in RANK−/− mice, although less than in WT. These results further indicate that osteoclasts are not strictly required for PTH anabolism, which presumably still occurs via stimulation of modeling-based bone formation. However the magnitude of PTH anabolic effects on the skeleton, in particular its additive effects with antiresorptives, depends on the extent of the remodeling space, as determined by the number and activity of osteoclasts on bone surfaces.

The transcription factor Sox4 is vital for fetal development, as Sox4–/– homozygotes die in utero. Sox4 mRNA is expressed in the early embryonic growth plate and is regulated by parathyroid hormone, but its function in bone modeling/remodeling is unknown. We report that Sox4+/– mice exhibit significantly lower bone mass (by dual-energy X-ray absorptiometry) from an early age, and fail to obtain the peak bone mass of wild-type (WT) animals. Microcomputed tomography (μCT), histomorphometry and biomechanical testing of Sox4+/– bones show reduced trabecular and cortical thickness, growth plate width, ultimate force and stiffness compared with WT. Bone formation rate (BFR) in 3-month-old Sox4+/– mice is 64% lower than in WT. Primary calvarial osteoblasts from Sox4+/– mice demonstrate markedly inhibited proliferation, differentiation and mineralization. In these cultures, osterix (Osx) and osteocalcin (OCN) mRNA expression was reduced, whereas Runx2 mRNA was unaffected. No functional defects were found in osteoclasts. Silencing of Sox4 by siRNA in WT osteoblasts replicated the defects observed in Sox4+/– cells. We demonstrate inhibited formation and altered microarchitecture of bone in Sox4+/– mice versus WT, without apparent defects in bone resorption. Our results implicate the transcription factor Sox4 in regulation of bone formation, by acting upstream of Osx and independent of Runx2.

This work provides a systematic review of the literature from January 2003 to April 2014 pertaining to the incidence, pathophysiology, diagnosis, and treatment of osteonecrosis of the jaw (ONJ), and offers recommendations for its management based on multidisciplinary international consensus. ONJ is associated with oncology-dose parenteral antiresorptive therapy of bisphosphonates (BP) and denosumab (Dmab). The incidence of ONJ is greatest in the oncology patient population (1% to 15%), where high doses of these medications are used at frequent intervals. In the osteoporosis patient population, the incidence of ONJ is estimated at 0.001% to 0.01%, marginally higher than the incidence in the general population (

Intermittent administration of parathyroid hormone (PTH) induces bone remodeling and renewed bone modeling, resulting in net bone gain. beta-blockers improve trabecular bone architecture in young ovariectomized mice by preventing the inhibition of bone formation and stimulation of bone resorption induced by the adrenergic system. To test the hypothesis that PTH and beta-blockers may exert synergistic effects on the skeleton, 15-week-old ovariectomized mice were either given oral propranolol (PRO) or left untreated for 8 weeks, adding daily hPTH(1-34) (80 microg/kg/day) or vehicle (VEH) during the last 4 weeks. The skeletal response was evaluated using pDXA, microCT, histomorphometry and biochemical markers. PRO significantly attenuated loss of bone mineral density (BMD) at whole body (WB) (-0.1% in PRO vs. -2.4% in VEH, P < 0.05), but not at spine or femur 4 weeks after OVX. Thereafter, PTH increased BMD at all sites in both PRO- and VEH-treated mice (+6.7% to +14%, P < 0.05 to P < 0.0001 vs. VEH). Over 8 weeks, sequential-combined treatment of PRO and PTH significantly improved BMD over PTH alone at WB (+9.1% vs. +4.4% over baseline, respectively, P < 0.005) and spine (+9% vs. -1.7%, respectively, P < 0.05). These effects were paralleled by a decrease in TRACP5b with PRO (P < 0.05 vs. VEH) and an increase in osteocalcin with PTH, irrespective of PRO (P < 0.0001 vs. VEH). Trabecular bone microarchitecture, such as BV/TV, trabecular number and ConnD, was significantly improved by sequential-combined treatment of PRO and PTH compared to PTH alone. At midshaft femur, both PRO and PTH significantly increased cross-sectional area (CSA), but the effects of the two drugs on CSA and cortical thickness were not additive. Dynamic histomorphometry indicated that bone formation was increased by PTH at both cortical and trabecular surfaces, whereas PRO increased osteoblast number and surface on trabecular surfaces. The combined treatment further improved the extent of mineralization and BFR over PTH alone (P < 0.05) at endocortical surfaces and recapitulated the effects of PTH and PRO alone on trabecular surfaces. These results indicate that beta-adrenergic blockade may partially improve the bone remodeling balance induced by estrogen deficiency. In turn, PRO exerted synergistic effects with intermittent PTH on bone mass and cancellous bone architecture. As such, combined therapy of beta-blockers and PTH may be of interest in the treatment of postmenopausal osteoporosis.

Intermittent PTH administration increases bone turnover, resulting in net anabolic effects on bone. These effects are primarily mediated by intracellular cAMP signaling. However, the molecular mechanisms that regulate PTH activity in bone remain incompletely understood. β-Arrestin2, a G protein-coupled receptor regulatory protein, inhibits PTH-stimulated cAMP accumulation in vitro. Using β-arrestin2−/− (KO) and wild-type (WT) mice, we investigated the response to PTH in primary osteoblasts (POB) and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo. Compared with that in WT mice, PTH-stimulated intracellular cAMP was increased and sustained in KO POB. Intermittent exposure of POB to PTH significantly decreased the ratio of osteoprotegerin (OPG) receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression in KO POB, whereas it increased this ratio in WT POB. Total body bone mass and cortical and trabecular bone parameters were 5–10% lower in male KO mice compared with WT, and these differences were magnified upon in vivo administration of intermittent PTH (80 μg/kg·d) for 1 month. Thus, PTH significantly increased total body bone mineral content as well as vertebral trabecular bone volume and thickness in WT, but not KO mice. The anabolic response to PTH in cortical bone was also slightly more pronounced in WT than KO mice. Histomorphometry indicated that PTH prominently stimulated indexes of bone formation in both WT and KO mice, whereas it significantly increased indexes of bone resorption (i.e. osteoclast number and surface) in KO mice only. In conclusion, these results suggest that β-arrestins may specify the activity of intermittent PTH on the skeleton by limiting PTH-induced osteoclastogenesis.

High-fat diet-induced obesity (DIO) in rodents is associated with hyperleptinemia and resistance to leptin, but the response to agents acting downstream of leptin receptors remains unknown. We assessed the response of mice with DIO to treatment with MTII, an α-melanocyte-stimulating hormone analog. MTII delivered four times daily by intraperitoneal injection to C57BL/6J mice produced a dose-responsive effect on food intake, body weight, leptin, corticosterone, insulin, and free fatty acids. In DIO mice, administration of MTII 100 μg q.i.d. i.p. markedly suppressed feeding during the first 4 days of treatment, with food intake returning to control levels at day 5. Progressive weight loss also occurred over the first 4 days, after which weight plateaued at a level below control. After 8 days of treatment, MTII-treated DIO mice had major suppression of both leptin and insulin levels. Central administration of MTII for 4 days (10 nmol/day) in DIO mice significantly suppressed food intake, induced weight loss, and increased energy expenditure. These results indicate that 1) MTII administration to DIO mice causes suppression of food intake and body weight loss, and decreased food intake is primarily responsible for weight loss; 2) peripheral MTII improves insulin resistance in DIO mice; 3) “tachyphylaxis” to the effect of chronic MTII treatment on food intake occurs; and 4) at least some of the effects of MTII are exerted centrally. In conclusion, treatment with a melanocortin agonist is a promising therapeutic approach to DIO and associated insulin resistance.

In many species, delayed sexual maturation occurs when metabolic conditions are not satisfactory. Recently, leptin was shown to be involved in the regulation of food intake and body mass. Furthermore, leptin administration was shown to advance sexual maturation in mice and to rescue sexual function in adverse metabolic conditions. We examined plasma leptin levels in female rats during development and evaluated the role of leptin on sexual maturation in rats subjected to food restriction. In normal rats, plasma leptin levels were low at day 24 of life, then steadily increased during the juvenile period, reaching 740+/-56 pg/ml at 40 days at time of vaginal opening (VO) and further increasing by day 60 (957+/-73 pg/ml). Food restriction initiated at day 25 strongly impaired this increase, in proportion to the severity of the restriction. With a daily food intake reduced to 7-8 g/day, that permanently prevented VO, plasma leptin levels were very low at day 53 (169+/-67 pg/ml). Following switch to ad libitum feeding, plasma leptin reached high levels within 2 days (1577+/-123 pg/ml), and VO occurred 4 days later. If the severe food restriction was maintained and a central infusion of leptin (10 microg/day) was initiated, a significant decrease in body weight compared with vehicle-infused controls was observed. In these conditions, VO occurred in eight out of the nine leptin-treated rats, representing induction of the process of sexual maturation confirmed by increases in ovarian and uterine weights. This induction of sexual maturation exclusively results from a central effect of leptin because no leak of the i.c.v. administered leptin to the general circulation was observed. These data suggest that the rising plasma levels of leptin in the prepubertal period represent a signal to the brain indicating that the young animal is metabolically ready to go through the process of sexual maturation.

Neuropeptide Y (NPY) is involved in the central regulation of appetite, sexual behavior, and reproductive function. We have previously shown that chronic infusion of NPY into the lateral ventricle of normal rats produced an obesity syndrome characterized by hyperphagia, hyperinsulinism and collapse of reproductive function. We further demonstrated that acute inhibition of LH secretion in castrated rats was preferentially mediated by the NPY receptor subtype 5 (Y(5)). In the present study, the effects of chronic, central infusion of NPY, or the mixed Y2-Y5 agonist PYY(3-36), were evaluated both in normal male C57BL/6J mice and Sprague-Dawley rats. After a 7-day infusion to male mice, both NPY and PYY(3-36) at 5 nmol per day, induced marked hyperphagia leading to significant increases in body and fat pad weights. Furthermore, both compounds markedly reduced several markers of the reproductive axis. In the rat study, PYY(3-36) was more active than NPY to inhibit the pituitary-testicular axis, confirming the importance of the Y5 subtype for such effects. In the mouse, chronic NPY infusion induced a sustained increase in corticosterone and insulin secretion. Plasma leptin levels were also markedly increased possibly explaining the observed reduction in gene expression for hypothalamic NPY. Gene expression for hypothalamic POMC was reduced in the NPY- or PYY(3-36)-infused mice, suggesting that NPY exacerbated food intake by both acting through its own receptor(s), and reducing the satiety signal driven by the POMC-derived alpha-MSH. The present study in the mouse suggests in analogy with available rat data, that constant exposure to elevated NPY in the hypothalamic area unabatedly enhances food intake leading to an obesity syndrome including increased adiposity, insulin resistance, hypercorticism, and hypogonadism, reminiscent of the phenotype of the ob/ob mouse, that displays elevated hypothalamic NPY secondary to lack of leptin negative feedback action.

Obesity in humans and in rodents is usually associated with high circulating leptin levels and leptin resistance. To examine the molecular basis for leptin resistance, we determined the ability of leptin to induce hypothalamic STAT3 (signal transducer and activator of transcription) signaling in C57BL/6J mice fed either low-fat or high-fat diets. In mice fed the low-fat diet, leptin activated STAT3 signaling when administered via the intraperitoneal (ip) or the intracerebroventricular (icv) route, with the half-maximal dose being 30-fold less when given by the icv route. The high-fat diet increased body-weight gain and plasma leptin levels. After 4 weeks on the diet, hypothalamic STAT3 signaling after ip leptin administration was equivalent in both diet groups. In contrast, peripherally administered leptin was completely unable to activate hypothalamic STAT3 signaling, as measured by gel shift assay after 15 weeks of high-fat diet. Despite the absence of detectable signaling after peripheral leptin at 15 weeks, the mice fed the high-fat diet retained the capacity to respond to icv leptin, although the magnitude of STAT3 activation was substantially reduced. These results suggest that leptin resistance induced by a high-fat diet evolves during the course of the diet and has at least two independent causes: an apparent defect in access to sites of action in the hypothalamus that markedly limits the ability of peripheral leptin to activate hypothalamic STAT signaling, and an intracellular signaling defect in leptin-responsive hypothalamic neurons that lies upstream of STAT3 activation.

A number of studies have indicated that neuropeptide Y (NPY) is a central regulator of the gonadotropic axis, and the Y1 receptor was initially suggested to be implicated. As at least five different NPY receptor subtypes have now been characterized, the aim of the present study was to reinvestigate the pharmacological profile of the receptor(s) mediating the inhibitory action of NPY on LH secretion by using a panel of NPY analogs with different selectivity toward the five NPY receptor subtypes. When given intracerebroventricularly (icv) to castrated rats, a bolus injection of native NPY (0.7–2.3 nmol) dose-dependently decreased plasma LH. Peptide YY (PYY; 2.3 nmol) was as potent as NPY, suggesting that the Y3 receptor is not implicated. Confirming previous data, the mixed Y1, Y4, and Y5 agonist[ Leu31,Pro34]NPY (0.7–2.3 nmol) inhibited LH release with potency and efficacy equal to those of NPY. Neither the selective Y2 agonist C2-NPY (2.3 nmol) nor the selective Y4 agonist rat pancreatic polypeptide affected plasma LH, excluding Y2 and Y4 subtypes for the action of NPY on LH secretion. The mixed Y4-Y5 agonist human pancreatic polypeptide (0.7–7 nmol) as well as the mixed Y2-Y5 agonist PYY3–36 (0.7–7 nmol) that displayed very low affinity for the Y1 receptor, thus practically representing selective Y5 agonists in this system, decreased plasma LH with potency and efficacy similar to those of NPY, indicating that the Y5 receptor is mainly involved in this inhibitory action of NPY on LH secretion.[ d-Trp32]NPY, a selective, but weak, Y5 agonist, also inhibited plasma LH at a dose of 7 nmol. Furthermore, the inhibitory action of NPY (0.7 nmol) on LH secretion could be fully prevented, in a dose-dependent manner (6–100 μg, icv), by a nonpeptidic Y5 receptor antagonist. This antagonist (60 μg, icv) also inhibited the stimulatory action of NPY (0.7 nmol) on food intake. The selectivity of PYY3–36, human PP,[ d-Trp32]NPY, and the Y5 antagonist for the Y5 receptor subtype was further confirmed by their ability to inhibit the specific[ 125I][Leu31,Pro34]PYY binding to rat brain membrane homogenates in the presence of the Y1 receptor antagonist BIBP3226, a binding assay system that was described as being highly specific for Y5-like receptors. With the exception of[ d-Trp32]NPY, all analogs able to inhibit LH secretion were also able to stimulate food intake. Taken together, these results indicate that the Y5 receptor is involved in the negative control by NPY of the gonadotropic axis.

The pulsatile luteinizing hormone (LH) and testosterone secretions were studied during serial blood collections performed at 7-min time intervals in the male rat. In fed rats, a discontinuous pattern of LH secretion was observed. Periods without secretion alternated with active secretory episodes consisting in trains of three to four LH peaks that triggered testosterone secretion usually 1–2 h later. The magnitude of the testosterone response was not correlated with the amplitude of the LH peaks. Isolated, single peaks of LH did not evoke clear testosterone responses. Forty-eight hours after initiation of fasting, testosterone secretion was markedly decreased, but integrated LH secretion was only partly reduced. Chronic infusion of neuropeptide Y (NPY; 18 μg/day, icv) reduced testosterone secretion to very low levels and abolished pulsatile LH secretion or testosterone response to isolated LH peaks. In conclusion, the stimulation of testosterone secretion by LH necessitates several LH peaks organized in a proper sequence, and the testosterone response is not immediate. Low testosterone secretion in fasting rats appears to result from disappearance of coordinated, multiple LH peaks of sufficient size. Inhibition of the gonadotropic axis achieved by central NPY administration is due to either absence of LH peak “clusters” or occurrence of nonfunctional single LH peaks.

The ability of hCG and LH to induce testosterone (T) secretion by Leydig cells is well documented. However, the influence of the pulsatile nature of LH secretion, with varying frequency and amplitude, on T production in vivo is less clear. In our earlier studies on the relationship between pulsatile LH release and T secretion in adult male rats, no simple causality was observed. The recent availability of rat recombinant (rec) LH prompted us to study the effects of one and of three i.v. pulses of different doses of rat recLH on T secretion in adult male rats rendered hypogonadotropic by treatment with the GnRH antagonist cetrorelix. One or three supraphysiological pulses of 1.0 microg of rat recLH produced similar maximal T responses. In contrast, high physiological LH pulses (0.1 microg) produced discrete T-response peaks, whereas multiple low pulses of LH (0.03 microg) were needed before a T response was achieved. The T stimulation was greatly diminished after an LH pulse of 0.1 microg if rats had been treated on the previous day with pulses of 0.03 vs. 0.1 microg rat recLH, apparently because of prolonged LH deprivation and lack of Leydig cell priming due to the GnRH antagonist treatment. The novel preparation of rat recLH provides a physiologically relevant tool for studying the complex relationship between pulsatile LH release and T secretion in male rats.

The discovery of leptin has generated an extraordinary interest in the field of obesity but also in the understanding of the relationship between metabolic status and the neuroendocrine system. Following the initial demonstration that leptin administration to fasting mice can 'protect' neuroendocrine secretions and prevent the changes that are associated with fasting, the concept has emerged that a normal leptin secretion is a prerequisite for normal neuroendocrine secretions. Several unfavorable metabolic situations are associated with low plasma leptin, increased secretion of hypothalmic neuropeptide Y (NPY), and hypogonadism, and a causal relationship has been evoked. Severe dietary restriction in juvenile female rats is associated with low plasma leptin and sexual immaturity. Cessation of food restriction leads to immediate increase in plasma leptin followed 4 days later by vaginal opening. If food restriction is maintained, central leptin infusion can induce sexual maturation, thus demonstrating that leptin can act as a signal for the onset of puberty. In untreated type-I diabetic rats, hypogonadism is associated with very low plasma leptin and increased hypothalmic NYP synthesis and oestrous cyclicity. Fasting rapidly inhibits growth hormone (GH) secretion in association with low plasma leptin and elevated hypothalmic NPY. Central infusion of leptin to fasting rats was able to completely prevent the collapse of GH secretion and to maintain a normal low NPY synthesis. In summary, normally elevated plasma levels appear to be a prerequisite for normal GH and gonadotropin secretion in the rat. Degradation of metabolic conditions results in a rapid reduction of circulating leptin that could represent the signal for several alterations of neuroendocrine secretions. At the level of the hypothalamus, leptin could act on NPY neurons to transduce part or all of this 'metabolic' message. The possibility that changing plasma levels for leptin also affect peripheral endocrine targets, such as pituitary, ovary, adrenal or pancreas, is likely since these endocrine organs express functional long-term leptin receptors.

The obese gene (OB) product, leptin, has been shown to exert control on metabolic processes such as food intake and body weight homeostasis, possibly through a neuropeptide Y (NPY) neurotransmission. More recently, leptin has been shown to control several neuroendocrine axes, modulating pituitary hormone secretions in function of metabolic conditions. Since in the rat growth hormone (GH) secretion is dependent upon prevailing metabolic conditions, and NPY has been shown to be implicated in the feedback mechanisms of this hormone, we reasoned that leptin could also exert control over GH secretion and we examined this hypothesis in male rats submitted to a 3-day fast. Circulating leptin concentrations measured by RIA abruptly fell to low values after 24 h of fasting and remained low thereafter. Upon refeeding, leptin secretion regularly increased. As shown by others, pulsatile GH secretion had disappeared after 3 days of fasting. Centrally administered leptin (10 µg/day, i.c.v. infusion initiated at the beginning of the fasting period) totally prevented the disappearance of pulsatile GH secretion. No leak of centrally administered leptin to the general circulation was observed. Infusing the same amount of leptin intracerebroventricularly to rats receiving ad libitum feeding produced a severe reduction in food intake but maintained a normal GH secretory pattern. In contrast, pair-fed rats, submitted to the same food restriction, exhibited a marked reduction in GH secretion. Hypothalamic NPY gene expression, estimated by Northern blot analysis, was significantly increased in fasting rats, and decreased in leptin-treated, fasting rats. In rats receiving ad libitum feeding, leptin treatment reduced NPY gene expression, consistent with the observed reduction in food intake, whereas pair-fed animals logically exhibited increased NPY gene expression. In both situations with reduced feeding, normal GH secretion was seen in leptin-treated animals exhibiting low NPY gene expression, whereas decreased or abolished GH secretion was seen in animals not receiving leptin and exhibiting increased NPY mRNA levels. Interestingly, despite maintenance of normal GH secretion in leptin-treated, fasting rats, plasma IGF-I levels were low, as in vehicle-treated rats. Indeed, hepatic gene expression for both GH receptor and IGF-I was markedly reduced by fasting, and no correction was seen with leptin treatment. In summary, the regulation of GH secretion, at least the changes linked with malnutrition, appears to be dependent upon a leptin signal, perceived centrally, possibly related to circulating levels of this new hormone. The present data suggest that leptin can rescue normal pulsatile GH secretion by preventing the documented inhibitory action of NPY on GH secretion.

The role of neuropeptide Y (NPY) in the regulation of sexual function is still controversial. Whereas central NPY administration is stimulatory during proestrus in the rat and other species, such administration is inhibitory in castrated animals and we have shown that chronic, central infusion of NPY inhibits both the gonadotropic and somatotropic axis in adult rats. Previous studies have suggested that NPY could be involved in the triggering of the first ovulatory LH surge and a recent report indicated that a single central NPY injection given at 30 days of life could advance sexual maturation. We therefore evaluated the effect of chronic NPY administration on the timing of sexual maturation in the female rat and compared the effects obtained with those induced by a single NPY injection. Constant infusion of NPY (18 µg/day) into the lateral ventricle delayed sexual maturation as seen by the absence of vaginal opening, delayed ovarian growth and reduced number of pituitary GnRH receptors. This inhibitory effect was seen as long as NPY was infused. In contrast, a single NPY injection either at 30 days of life or earlier, or repeated single injections between 28 and 31 days of life, did not modify the pace of sexual maturation. Unlike what is observed in adult animals, overall food intake was only minimally increased between 30 and 37 days of life in chronically NPY-infused rats, but this increase became more important thereafter. In acutely NPY-treated animals, the expected brisk acceleration of food intake in the 3 h following central NPY administration took place, and the amplitude of this acceleration increased significantly between 28 and 31 days of life. Plasma insulin-like growth factor levels were markedly reduced in all chronically NPY-treated rats, probably reflecting a decrease in GH secretion. These data demonstrate that elevated NPY levels in the hypothalamus can delay sexual maturation in normal female rats. The exact mechanism of action of NPY for this inhibitory action is still unclear: NPY could either display a specific inhibitory action on GnRH release or down-regulate NPY receptors involved in the mediation of the stimulatory action of NPY on the LH ovulatory surge in the rat.

Inflammatory bowel diseases are commonly complicated by weight and bone loss. We hypothesized that IL-15, a pro-inflammatory cytokine expressed in colitis and an osteoclastogenic factor, could play a central role in systemic and skeletal complications of inflammatory bowel diseases. We evaluated the effects of an IL-15 antagonist, CRB-15, in mice with chronic colitis induced by oral 2% dextran sulfate sodium for 1 week, followed by another 1% for 2 weeks. During the last 2 weeks, mice were treated daily with CRB-15 or an IgG2a control antibody. Intestinal inflammation, disease severity, and bone parameters were evaluated at days 14 and 21. CRB-15 improved survival, early weight loss, and colitis clinical score, although colon damage and inflammation were prevented in only half the survivors. CRB-15 also delayed loss of femur bone mineral density and trabecular microarchitecture. Bone loss was characterized by decreased bone formation, but increased bone marrow osteoclast progenitors and osteoclast numbers on bone surfaces. CRB-15 prevented the suppression of osteoblastic markers of bone formation, and reduced osteoclast progenitors at day 14, but not later. However, by day 21, CRB-15 decreased tumor necrosis factor α and increased IL-10 expression in bone, paralleling a reduction of osteoclasts. These results delineate the role of IL-15 on the systemic and skeletal manifestations of chronic colitis and provide a proof-of-concept for future therapeutic developments.

Neuropeptide Y (NPY) is known to be involved in the central regulation of appetite, sexual behavior, and reproductive functions. Whereas central administration of NPY strongly stimulates feeding in satiated animals, diet restriction produces overexpression of NPY in the arcuate and paraventricular nuclei that might reflect behavioral adaptations to shortage of food. Previous studies indicated that central administration of NPY resulted in controversial actions on LH secretion, either stimulatory or inhibitory. In order to analyze the chronic effect on pituitary function of centrally administered NPY, stainless steel cannulae were implanted in the right lateral ventricles of intact 45-day-old Sprague-Dawley female rats. Ten days later, Alzet osmotic minipumps filled with saline or different concentrations of NPY adjusted to deliver 3, 6, 12, or 18 micrograms/day were connected to the intracerebroventricular (icv) cannulae, implanted sc dorsally, and the effects of these treatments evaluated after 7 days. Chronic icv infusion of NPY produced the expected dose-related increase in food intake [25.3 +/- 0.8 g/day (basal) to 47.9 +/- 4.3 g/day (highest NPY dose)] and body wt gain (3.7 +/- 0.4-11.5 +/- 1.4 g/day). Basal insulinemia was highly correlated to the increase in food intake. This orexigenic action of NPY was accompanied by a drastic dose-related decrease in pituitary wt (14.0 +/- 0.5-8.3 +/- 0.3 mg), pituitary concentration of GnRH receptors, a known marker of the activity of the hypothalamo-pituitary gonadal axis (15.2 +/- 1.7-5.2 +/- 0.5 fmol/mg), and ovarian wt (84.0 +/- 4.2-49 +/- 6.7 mg). Ovulation was impaired in NPY-treated animals as seen by daily inspection of vaginal smears. A sharp dose-dependent decrease in plasma levels of insulin-like growth factor I was also observed [934 +/- 64 ng/ml (basal) to 385 +/- 26 ng/ml (highest NPY dose)], probably secondary to a decrease in GH secretion. Whereas these data confirm the central action of NPY to stimulate appetite in satiated animals, they provide the first demonstration that chronic icv administration of NPY unequivocally inhibits gonadotropin secretion and sexual function in intact female rats. These data also confirm that NPY can suppress GH secretion and other anabolic hormones. In conclusion, these results may indicate a physiological role of NPY as an integrator of different adaptive behaviors in periods of unfavorable metabolic conditions such as diet restriction, extending its action to inhibition of sexual functions and anabolic processes.

8,8''-Biapigeninyl (BA), a condensation product of two apigenin molecules, is found abundantly in the nuts of Cupressus sempervirens. We investigated the effects of BA on murine bone cells in vitro and in ovariectomized (OVx) mice. BA at 10(-10)M and 10(-8)M, inhibited osteoclastogenesis of bone marrow cells (BMCs) and displayed concentration dependence. BA at 10(-8) M and 10(-6) M inhibited differentiation of 3T3-L1 and BMCs to mature adipocytes. BA (10(-10)M) stimulated osteoblast proliferation, differentiation and mineralization. In stimulating osteoblast function, BA was found to be 10(4)-fold more potent than apigenin. The effect of BA in osteoblasts appeared to be mediated via estrogen receptors (ER) as antiestrogen, ICI-182780 abolished BA-stimulated osteoblast differentiation. In OVx mice BA treatment (at 1.0-, 5.0- and 10.0 mg kg(-1) day(-1) doses) given orally for 30 days dose-dependently inhibited mRNA levels of osteoclastic genes including tartrate-resistant acid phosphatase, receptor activator of nuclear factor (RANK), tumor necrosis factor alpha, interleukin-6 and the ratio of RANK ligand/osteoprotegerin ratio in bones compared with OVx mice treated with vehicle. In addition, BA treatment to OVx mice dose-dependently stimulated production of osteoprogenitor cells in the bone marrow and increased mRNA levels of osteogenic genes core binding factor alpha-1, type I collagen and bone morphogenic protein-2 in bones compared with OVx+vehicle group. Microcomputed tomography revealed that BA treatment to OVx mice improved parameters of trabecular and cortical architecture. BA exhibited no uterine estrogenicity. From these data, we conclude that BA exerts osteoprotective effect in OVx mice by multiple beneficial effects on bone cells.

OBJECTIVE: T cell production of RANKL, interferon-gamma (IFNgamma), and other cytokines in inflammatory processes such as rheumatoid arthritis or secondary to conditions such as estrogen deficiency stimulates osteoclast activity, which leads to bone resorption and bone loss. The purpose of this study was to characterize the effects of interleukin-15 (IL-15), a master T cell growth factor whose role in bone remodeling remains unknown. METHODS: We used mice lacking the IL-15 receptor (IL-15Ralpha(-/-) ) to investigate the effects of IL-15 on osteoclast development, T cell and dendritic cell activation in vitro and in vivo, bone mass, and microarchitecture in intact and ovariectomized (OVX) mice. RESULTS: In wild-type (WT) animals, IL-15 and RANKL provided a costimulatory signal for osteoclast development. Spleens from IL-15Ralpha(-/-) mice contained few c-Kit+ osteoclast precursors, and the expression of NF-ATc1 and the osteoclastogenic response to RANKL were impaired. In addition, dendritic cell-dependent and T cell-dependent mechanisms of osteoclast activation, including RANKL and IFNgamma production, were impaired in IL-15Ralpha(-/-) mice. In turn, IL-15Ralpha(-/-) T cells failed to stimulate WT osteoclasts, whereas WT T cells failed to stimulate IL-15Ralpha(-/-) osteoclasts. Compared with WT mice, both intact and OVX IL-15Ralpha(-/-) mice had significantly greater bone mineral density and microarchitecture, including a higher trabecular bone volume fraction and cortical thickness. The numbers of osteoclasts on the bone surface as well as markers of bone turnover were significantly decreased in IL-15Ralpha(-/-) mice. CONCLUSION: In the absence of IL-15 signaling, several converging mechanisms of osteoclastogenesis are inhibited, both directly and indirectly, through T cells, which leads to a high bone mass phenotype. Targeting the IL-15 pathway may represent a novel therapeutic approach to treating primary and secondary osteoporosis.

Changes in whole body energy levels are closely linked to alterations in body weight and bone mass. Here, we show that hypothalamic signals contribute to the regulation of bone mass in a manner consistent with the central perception of energy status. Mice lacking neuropeptide Y (NPY), a well-known orexigenic factor whose hypothalamic expression is increased in fasting, have significantly increased bone mass in association with enhanced osteoblast activity and elevated expression of bone osteogenic transcription factors, Runx2 and Osterix. In contrast, wild type and NPY knockout (NPY (-/-)) mice in which NPY is specifically over expressed in the hypothalamus (AAV-NPY+) show a significant reduction in bone mass despite developing an obese phenotype. The AAV-NPY+ induced loss of bone mass is consistent with models known to mimic the central effects of fasting, which also show increased hypothalamic NPY levels. Thus these data indicate that, in addition to well characterized responses to body mass, skeletal tissue also responds to the perception of nutritional status by the hypothalamus independently of body weight. In addition, the reduction in bone mass by AAV NPY+ administration does not completely correct the high bone mass phenotype of NPY (-/-) mice, indicating the possibility that peripheral NPY may also be an important regulator of bone mass. Indeed, we demonstrate the expression of NPY specifically in osteoblasts. In conclusion, these data identifies NPY as a critical integrator of bone homeostatic signals; increasing bone mass during times of obesity when hypothalamic NPY expression levels are low and reducing bone formation to conserve energy under 'starving' conditions, when hypothalamic NPY expression levels are high.

PTH-stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule beta-arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in beta-arrestin2(-/-) mice and suggested that beta-arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of beta-arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and beta-arrestin2(-/-) mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH-stimulated OCs was higher in BM cultures from beta-arrestin2(-/-) compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in beta-arrestin2(-/-) compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in beta-arrestin2(-/-). PTH downregulated Efn and Eph genes in beta-arrestin2(-/-), but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in beta-arrestin2(-/-) compared with WT. Histomorphometry showed that OC number was higher in OVX-beta-arrestin2(-/-) compared with WT. These results indicate that beta-arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, beta-arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.

Activation of β2-adrenergic receptors inhibits osteoblastic bone formation and enhances osteoclastic bone resorption. Whether β-blockers inhibit ovariectomy-induced bone loss and decrease fracture risk remains controversial. To further explore the role of β-adrenergic signaling in skeletal acquisition and response to estrogen deficiency, we evaluated mice lacking the three known β-adrenergic receptors (β-less). Body weight, percent fat, and bone mineral density were significantly higher in male β-less than wild-type (WT) mice, more so with increasing age. Consistent with their greater fat mass, serum leptin was significantly higher in β-less than WT mice. Mid-femoral cross-sectional area and cortical thickness were significantly higher in adult β-less than WT mice, as were femoral biomechanical properties (+28 to +49%, P < 0.01). Young male β-less had higher vertebral (1.3-fold) and distal femoral (3.5-fold) trabecular bone volume than WT (P < 0.001 for both) and lower osteoclast surface. With aging, these differences lessened, with histological evidence of increased osteoclast surface and decreased bone formation rate at the distal femur in β-less vs. WT mice. Serum tartrate-resistance alkaline phosphatase-5B was elevated in β-less compared with WT mice from 8–16 wk of age (P < 0.01). Ovariectomy inhibited bone mass gain and decreased trabecular bone volume/total volume similarly in β-less and WT mice. Altogether, these data indicate that absence of β-adrenergic signaling results in obesity and increased cortical bone mass in males but does not prevent deleterious effects of estrogen deficiency on trabecular bone microarchitecture. Our findings also suggest direct positive effects of weight and/or leptin on bone turnover and cortical bone structure, independent of adrenergic signaling. Mice lacking ß-adrenergic receptors have increased body weight, bone mineral density, and bone turnover versus controls, but are not protected from bone loss due to deficiency of estrogens..

Skeletal anabolism with PTH is achieved through daily injections that result in brief exposure to the peptide. We hypothesized that similar anabolic effects could be achieved with less frequent but more sustained exposures to PTH. A PTH-Fc fusion protein with a longer half-life than PTH(1-34) increased cortical and cancellous BMD and bone strength with once- or twice-weekly injections. Introduction: The anabolic effects of PTH are currently achieved with, and thought to require, daily injections that result in brief exposure to the peptide. We hypothesized that less frequent but more sustained exposures to PTH could also be anabolic for bone, provided that serum levels of PTH were not constant. Materials and Methods: PTH(1-34) was fused to the Fc fragment of human IgG1 to increase the half-life of PTH. Skeletal anabolism was examined in mice and rats treated once or twice per week with this PTH-Fc fusion protein. Results: PTH-Fc and PTH(1-34) had similar effects on PTH/PTHrP receptor activation, internalization, and signaling in vitro. However, PTH-Fc had a 33-fold longer mean residence time in the circulation of rats compared with that of PTH(1-34). Subcutaneous injection of PTH-Fc once or twice per week resulted in significant increases in bone volume, density, and strength in osteopenic ovariectomized mice and rats. These anabolic effects occurred in association with hypercalcemia and were significantly greater than those achiev- able with high concentrations of daily PTH(1-34). PTH-Fc also significantly improved cortical bone volume and density under conditions where daily PTH(1-34) did not. Antiresorptive co-therapy with estrogen further enhanced the ability of PTH-Fc to increase bone mass and strength in ovariectomized rats. Conclusions: These results challenge the notion that brief daily exposure to PTH is essential for its anabolic effects on cortical and cancellous bone. PTH-derived molecules with a sustained circulating half-life may represent a powerful and previously undefined anabolic regimen for cortical and cancellous bone. J Bone Miner Res 2007;22:1534-1547. Published online on June 18, 2007; doi: 10.1359/JBMR.070616

Cytoplasmic arrestins regulate PTH signaling in vitro. We show that female β-arrestin2−/− mice have decreased bone mass and altered bone architecture. The effects of intermittent PTH administration on bone microarchitecture differed in β-arrestin2−/− and wildtype mice. These data indicate that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH at endosteal and periosteal bone surfaces. Introduction: The effects of PTH differ at endosteal and periosteal surfaces, suggesting that PTH activity in these compartments may depend on some yet unidentified mechanism(s) of regulation. The action of PTH in bone is mediated primarily by intracellular cAMP, and the cytoplasmic molecule β-arrestin2 plays a central role in this signaling regulation. Thus, we hypothesized that arrestins would modulate the effects of PTH on bone in vivo. Materials and Methods: We used pDXA, μCT, histomorphometry, and serum markers of bone turnover to assess the skeletal response to intermittent PTH (0, 20, 40, or 80 μg/kg/day) in adult female mice null for β-arrestin2 (β-arr2−/−) and wildtype (WT) littermates (7-11/group). Results and Conclusions: β-arr2−/− mice had significantly lower total body BMD, trabecular bone volume fraction (BV/TV), and femoral cross-sectional area compared with WT. In WT females, PTH increased total body BMD, trabecular bone parameters, and cortical thickness, with a trend toward decreased midfemoral medullary area. In β-arr2−/− mice, PTH not only improved total body BMD, trabecular bone architecture, and cortical thickness, but also dose-dependently increased femoral cross-sectional area and medullary area. Histomorphometry showed that PTH-stimulated periosteal bone formation was 2-fold higher in β-arr2−/− compared with WT. Osteocalcin levels were significantly lower in β-arr2−/− mice, but increased dose-dependently with PTH in both β-arr2−/− and WT. In contrast, whereas the resorption marker TRACP5B increased dose-dependently in WT, 20-80 μg/kg/day of PTH was equipotent with regard to stimulation of TRACP5B in β-arr2−/−. In summary, β-arrestin2 plays an important role in bone mass acquisition and remodeling. In estrogen-replete female mice, the ability of intermittent PTH to stimulate periosteal bone apposition and endosteal resorption is inhibited by arrestins. We therefore infer that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH on cancellous and cortical bone.

The human PTH receptor type 2 (PTH2R) is activated by PTH and tuberoinfundibular peptide of 39 residues (TIP39), resulting in cAMP and intracellular Ca signaling. We now report that, despite these similarities, PTH and TIP39 elicit distinct responses from PTH2R. First, TIP39 induced beta-arrestin and protein kinase Cbeta mobilization and receptor internalization, whereas PTH did not. However, PTH stimulated trafficking of these molecules for a chimeric PTH2R containing the N terminus and third extracellular loop of PTH receptor type 1 (PTH1R). Second, whereas PTH-stimulated cAMP activity was brief and rapidly resensitized, the response to TIP39 was sustained and partly desensitized for a prolonged period. PTH2R desensitization was mediated by beta-arrestin interaction with the C terminus (amino acids 426-457) of PTH2R, whereas beta-arrestin mobilization had a minor influence on PTH2R internalization in response to TIP39, as shown with C terminus deletion mutants and/or dominant negative forms of beta-arrestin and dynamin. These data contrast with PTH1R, at which these dominant negative mutants markedly inhibited receptor internalization. Collectively, these results further highlight how specific interactions within the ligand-receptor bimolecular complex mediate distinct postactivation responses of class II G protein- coupled receptors and provide novel insights into the physiological regulation of PTH2R activity.

Leptin deficiency causes obesity in rodents and humans, but circulating levels of leptin are paradoxically elevated in obesity. The mechanisms underlying this leptin resistance are unknown, but may involve reduced leptin transport across the blood-brain barrier via short isoforms of the leptin receptor (Ob-R). Here, we first quantified short Ob-R mRNA expression in isolated rat cerebral microvessels constituting the blood-brain barrier and found that Ob-Ra and Ob-Rc mRNA were abundantly expressed in similar amounts. Second, brain uptake of leptin was reduced in mice lacking Ob-R. Third, brain uptake of leptin in New Zealand Obese mice, a strain that responds to central, but not peripheral, leptin, was reduced, suggesting that their obesity is at least partly due to deficient leptin transport into the brain. Fourth, brain uptake of leptin was significantly reduced in diet-induced obese mice. Neither New Zealand Obese mice nor diet-induced obese mice exhibited significant decreases in Ob-R mRNA expression in isolated cerebral microvessels. These data support the ideas that short isoforms of Ob-R are involved in brain uptake of leptin and that impaired blood-brain barrier function contributes to the pathogenesis of obesity. However, the mechanisms by which obesity-related deficits in brain uptake of leptin occur remain to be defined.

The elucidation of the ob/ob gene product, leptin, not only closed a gap in the understanding of the regulation of feeding but also offered a rationale for the humoral link between metabolic function and the regulation of hypothalamo-pituitary secretions. Absence of leptin production as seen in ob/ob mice or in some rare cases of human obesity not only leads to hyperphagia, reduced energy expenditure, and hypercorticism, but also, most importantly, to sterility. Partial or total leptin resistance by mutation of the leptin receptor leads to a similar phenotype, as seen in db/db mice, fa/fa Zucker rats and again in rare cases of human obesity. Common human obesity is characterized by partial resistance to clearly elevated circulating leptin levels. A key finding was the demonstration that treatment of sterile ob/ob mice with leptin could quickly restore fertility, implying that a minimum level of circulating leptin is necessary for positive stimulation of the gonadotropic axis, at least as a permissive factor. A similar situation was seen with the treatment of two patients with leptin deficiency. Furthermore, leptin was shown to modulate other hypothalamo-pituitary axes. A general finding has been that leptin administration to animals undergoing severe reduction in energetic supply could reverse the negative effects of such a metabolic insult on most endocrine axes, with striking effects on pulsatile growth hormone (GH) and LH secretions in the rat. Ingestive behavior is controlled by multiple interacting neuroendocrine circuits that are leptin-dependent, involving neuropeptides such as Neuropeptide Y (NPY), Melanocortins (MCs), AGRP, CART, GLP-1, Orexin, etc. Generally speaking, a deficit in feeding corresponds to centrally induced reduction in sexual function. NPY is primarily known to stimulate feeding but also to modulate sexual behavior and gonadotropin secretion. Apparently reproductive function is not dependent on hypothalamic α-MSH or CART action. The recently described natural ligand of the GHRP receptor, ghrelin, which is synthesized and release by the stomach, not only stimulates GH secretion, as predicted, but also regulates feeding by acting on hypothalamic neuropeptides such as NPY. Ghrelin action seems to represent a counterbalance of leptin action. Whereas leptin decreases feeding and can produce anorexia, ghrelin stimulates feeding and eventu- ally produces obesity. Thus a trend for coordinated neuroendocrine regulation of ingestive and reproductive behavior in response to metabolic and environmental changes is expected, with a clear integrative role for leptin and possibly insulin. Still, much remains to be learned about the mode of action of leptin to activate GnRH secretion and reproduction in mammals.

To determine whether leptin signal transduction is exerted directly upon insulin-sensitive tissues in vivo, we examined the ability of iv leptin to acutely stimulate phosphorylation of STAT3, STAT1, and MAPK, and activities of PI 3-kinase and Akt, in insulin-sensitive tissues of normal rats. Both leptin (1 mg/kg iv x 3 min) and insulin (10 U/kg iv x 3 min) stimulated tyrosine phosphorylation of STAT3 5.6- to 6.0-fold and of STAT1 4.0-fold in adipose tissue. Leptin tended to increase STAT3 phosphorylation in liver and muscle. Both hormones also increased MAPK phosphorylation: leptin increased it 3.2- to 3.8-fold in adipose tissue and liver, whereas insulin stimulated MAPK phosphorylation 5.0-fold in adipose tissue, 6.8-fold in liver, and 2.5-fold in muscle. Leptin was much less effective than insulin at stimulating IRS pathways. Leptin increased IRS-1-associated PI 3-kinase activity in adipose tissue only 2.0-fold (P0.01) compared with the 10-fold effect of insulin. IRS-2-associated PI 3-kinase activity was increased 1.7-fold (P0.01) by leptin in liver and 6-fold by insulin. Akt phosphorylation and activity were not changed by leptin but increased with insulin. Lower concentrations of leptin (10 and 50 microg/kg) also stimulated STAT3 phosphorylation in fat. These effects appear to be direct because 3 min after leptin intracerebroventricular injection, phosphorylation of STAT3, STAT1, and MAPK were not stimulated in hypothalamus or adipose tissue. Furthermore, leptin activated STAT3 and MAPK in adipose tissue explants ex vivo and in 3T3-L1 adipocytes. Leptin did not activate STAT3 or MAPK in adipose tissue of db/db mice. Thus, leptin rapidly activates signaling pathways directly at the level of insulin sensitive tissues through the long-form leptin receptor, and these pathways overlap with, but are distinct from, those engaged by insulin.

We have previously described the preparation, purification and partial characterization of recombinant (rec) forms of rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the present study, the special functional features of these hormones were studied further, in vitro and in vivo, and compared with human recLH and recFSH, as well as with human urinary choriongonadotropin (hCG) and rat pituitary LH (NIDDK-RP3). In radioreceptor assay, the affinity of hCG binding to rat testis membranes was 5-fold higher than that of human recLH and 100-fold higher than that of rat recLH. In in vitro bioassay, using dispersed adult mouse interstitial cells or a mouse Leydig tumor cell line (BLT-1), hCG and human recLH were 10- to 20-fold more potent than rat recLH. Correspondingly, rat pituitary LH was about 10-fold less potent than rat recLH, and evoked a maximum testosterone response that was about half of that elicited by the other LH/CG preparations. Rat recFSH was about 10-fold less potent than human recFSH in stimulating cAMP production of a mouse Sertoli cell line (MSC-1) expressing the recombinant rat FSH receptor. The circulating half-times (T1/2) of rat and human rec hormones were assessed after i.v. injections into adult male rats rendered gonadotropin-deficient by treatment with a gonadotropin-releasing hormone antagonist. A novel immunometric assay was used for the rat FSH measurements. In the one-component model the T1/2 values of rat and human recLH were 18.2 +/- 1.9 min (n = 7) and 44.6 +/- 3.1 min (n = 7) respectively and those of rat and human recFSH were 88.4 +/- 10.7 min (n = 6) and 55.0 +/- 4.2 min (n = 6) respectively; the two-component models revealed similar differences between the rec hormone preparations. Collectively, rat recLH was eliminated significantly faster from the circulation than human recLH (P < 0.0001). In contrast, the elimination of rat recFSH was significantly slower than that of human recFSH (P = 0.02). In conclusion, rat recFSH and rat recLH display lower biopotencies per unit mass than the respective human hormones in vitro, and also in vivo for LH. This is paralleled by shorter T1/2 of rat recLH than the respective human hormone in the circulation, whereas human recFSH has a shorter T1/2 than human FSH. The special functional features of the rat rec gonadotropins emphasize the use of these preparations on studies of gonadotropin function in the rat, an important animal model for reproductive physiology.

Neuropeptide Y (NPY) is known to be involved in the central regulation of appetite, sexual behavior, and reproductive function. Whereas central administration of NPY strongly stimulates feeding in satiated animals, diet restriction or other unfavorable metabolic situations, such as diabetes, produce enhanced NPY gene expression and NPY release in the hypothalamus. Numerous studies have indicated that acute central administration of NPY results in various actions on LH secretion in the rat, either stimulatory or inhibitory. We recently demonstrated that chronic infusion of NPY into the lateral ventricle of adult intact female rats profoundly inhibited both the gonadotropic and somatotropic axes, with disruption of estrous cyclicity. Furthermore, we showed that central chronic infusion of NPY delayed sexual maturation in female rats. To analyze the effects of the same type of chronic NPY treatment on the pituitary-testicular axis, 45-day-old Sprague-Dawley male rats were implanted with stainless steel cannulas in the right lateral ventricle. Ten days later, Alzet osmotic minipumps were filled with different NPY solutions, adjusted to deliver 6, 18, or 36 micrograms/day, connected to the intracerebroventricular (icv) cannula, and sc implanted dorsally. The effects of these treatments were evaluated over 7 days. In one case, rats were castrated 5 days after initiation of NPY treatment, and the effect of castration was evaluated 2 days later. Chronic icv infusion of NPY produced the expected dose-related increases in food intake from 33.0 +/- 0.9 (basal) to 53.4 +/- 3.3 g/day (18 micrograms NPY/day) and body weight gain (5.7 +/- 0.7 to 10.5 +/- 1.2 d/day). As in female rats, this orexigenic action of NPY resulted in a significant dose-related decrease in pituitary weight, from 12.4 +/- 0.7 to 9.9 +/- 0.4 mg. The 7-day NPY infusion produced highly significant decreases in seminal vesicle weight (853 +/- 77 to 230 +/- 31 mg) and testis weight (3.82 +/- 0.09 to 3.18 +/- 0.15 g; P = 0.003). Plasma levels of testosterone (231 +/- 71 to 48 +/- 13 ng/dl), LH (20.7 +/- 3.7 to 9.1 +/- 1.2 ng/ml), and FSH (282 +/- 17 to 190 +/- 18 ng/ml) were markedly decreased at the 18 micrograms/day dosage, as also demonstrated for the 36 micrograms/day dosage. None of these effects was observed if vehicle was infused into the lateral ventricle instead of the NPY solution. When bilateral orchidectomy was performed 5 days after initiation of the NPY infusion (18 micrograms/day), the immediate LH and FSH rises usually seen after castration were seriously blunted.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y (NPY) is known to be involved in the central regulation of appetite, sexual behavior and reproductive function. Whereas central administration of NPY strongly stimulates feeding, diet restriction produces overexpression of NPY in the arcuate and paraventricular nuclei that might reflect behavioral adaptations to shortage of food. The role of NPY for the regulation of sexual function is still controversial. Whereas NPY is stimulatory during proestrus in the rat, acute administration of NPY is inhibitory in castrated animals and we have shown that chronic administration of NPY inhibits both the gonadotropic and somatotropic axis in adult female rats. In order to further analyse the role of NPY during sexual maturation, a model of delayed sexual maturation imposed by food restriction and return to ad-libitum feeding was used. Young female rats were restricted to 7-8 g food daily starting at 24 days of life (d). This restriction completely prevented sexual maturation. At 50 d, ICV cannulas were placed and at 60 d, Alzet minipumps either delivering NPY (18 micrograms/day) or vehicle into the ICV cannula were implanted dorsally. At 61 d, rats were switched to ad-libitum feeding, a change that produced vaginal opening within 4 days in all vehicle-treated rats. In the rats receiving NPY, significantly increased food intake and weight gain were observed but only one out of the 9 rats studied experienced vaginal opening at 66 d, the other 8 animals remaining sexually immature at 67 d at sacrifice. Sexual immaturity of NPY-treated rats was further confirmed by decreased ovarian weight and reduced number of pituitary GnRH receptors. Plasma IGF-I levels were markedly reduced in NPY-treated rats. Since food restriction has been shown both to increase hypothalamic NPY and to reduce or inhibit sexual function, these data bring evidence for the first time that NPY could be involved in the inhibition of sexual maturation imposed by food restriction, since maintenance of elevated NPY levels in the hypothalamus did prolong this state of sexual immaturity despite restoration of normal food intake.

NPY is known to be involved in the central regulation of appetite, metabolic processes, and reproductive functions. We have demonstrated that central administration of NPY results in an inhibition of the gonadotropic axis in the rat. In the female rat, sexual maturation can be delayed by severe diet restriction and in this situation, NPY is increased in the paraventricular nucleus (PVN). In order to evluate a potential role of NPY for this delay, we attempted to prolong this period of sexual immaturity by infusing NPY centrally after restauration of normal feeding condition. Sexual maturation was prevented by maintaining food allowance at 5.5 g/day. At 50 days of life (d), a cannula was placed in the lateral ventricle and at 60 d, the animals were implanted with Alzet pumps delivering 18 μg NPY daily to the cannula, or vehicle. One day later, food restriction was discontinued. The switch to ad libitum feeding produced a rapid acceleration of growth rate and, in the animals receiving vehicle, the expected full sexual maturation after 2 to 5 days. Animals receiving NPY exhibited an even more important increase in weight gain, but only one out of 9 rats studied had experienced vaginal opening after 6 days, at the end of pump capacity. In another series, sexual immaturity could be prolonged for 13 days. Thus, since NPY is increased in the PVN of food-restricted rats and that administration of NPY could prolong the situation of sexual immaturity observed during food restriction, it is tempting to speculate that NPY is instrumental for inhibiting GnRH release and sexual maturation in unfavorable metabolic conditions.