Monocyte CD14 and HLA-DR expression increases with disease duration and severity in amyotrophic lateral sclerosis.

Objective: To investigate changes in immune markers and frequencies throughout disease progression in patients with amyotrophic lateral sclerosis (ALS). Methods: In this longitudinal study, serial blood samples were collected from 21 patients with ALS over a time period of up to 16 months. Flow cytometry was used to quantitate CD14, HLA-DR, and CD16 marker expression on monocyte subpopulations and neutrophils, as well as their cell population frequencies. A Generalized Estimating Equation model was used to assess the association between changes in these immune parameters and disease duration and the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R). Results: CD14 expression on monocyte subpopulations increased with both disease duration and a decrease in ALSFRS-R score in patients with ALS. HLA-DR expression on monocyte subpopulations also increased with disease severity and/or duration. The expression of CD16 did not change relative to disease duration or ALSFRS-R. Finally, patients had a reduction in non-classical monocytes and an increase in the classical to non-classical monocyte ratio throughout disease duration. Conclusion: The progressive immunological changes observed in this study provide further support that monocytes are implicated in ALS pathology. Monocytic CD14 and HLA-DR surface proteins may serve as a therapeutic target or criteria for the recruitment of patients with ALS into clinical trials for immunomodulatory therapies.

The role of lipids in the central nervous system and their pathological implications in amyotrophic lateral sclerosis.

Lipids play an important role in the central nervous system (CNS). They contribute to the structural integrity and physical characteristics of cell and organelle membranes, act as bioactive signalling molecules, and are utilised as fuel sources for mitochondrial metabolism. The intricate homeostatic mechanisms underpinning lipid handling and metabolism across two major CNS cell types; neurons and astrocytes, are integral for cellular health and maintenance. Here, we explore the various roles of lipids in these two cell types. Given that changes in lipid metabolism have been identified in a number of neurodegenerative diseases, we also discuss changes in lipid handling and utilisation in the context of amyotrophic lateral sclerosis (ALS), in order to identify key cellular processes affected by the disease, and inform future areas of research.

Restraint stress increases prolactin-mediated phosphorylation of signal transducer and activator of transcription 5 in the hypothalamus and adrenal cortex in the male mouse.

Prolactin is a pleiotropic peptide hormone produced by the lactotrophs in the anterior pituitary. Its rate of secretion is primarily regulated by a negative-feedback mechanism where prolactin stimulates the activity of the tuberoinfundibular dopaminergic (TIDA) neurones, increasing their release of dopamine, which accesses the pituitary via the median eminence to suppress further prolactin secretion. In addition to its well established role in lactation, circulating prolactin is secreted in response to stress, although the mechanism by which this is achieved or its cellular targets remains unknown. In the present study, we show that 15 minutes of restraint stress causes an approximately seven-fold increase in circulating prolactin concentration in male mice. Monitoring prolactin receptor activation, using immunohistochemistry to determine the level and distribution of tyrosine phosphorylated signal transducer and activator of transcription 5 (pSTAT5), we show that this stress-induced increase in prolactin interacts with both central and peripheral targets. Restraint stress for 15 minutes significantly increased pSTAT5 staining in the arcuate nucleus, median eminence and the zona fasciculata of the adrenal cortex. In each case, this response was prevented by pretreating the animals with bromocriptine to block prolactin secretion from the pituitary. Interestingly, in contrast to many cells in the arcuate nucleus, stress reduced pSTAT5 staining of the TIDA neurones (identified by dual-labelling for tyrosine hydroxylase). This suggests that there is reduced prolactin signalling in these cells and thus potentially a decline in their inhibitory influence on prolactin secretion. These results provide evidence that prolactin secreted in response to acute stress is sufficient to activate prolactin receptors in selected target tissues known to be involved in the physiological adaptation to stress.

Hyperphagia in male melanocortin 4 receptor deficient mice promotes growth independently of growth hormone.

KEY POINTS: Loss of function of the melanocortin 4 receptor (MC4R) results in hyperphagia, obesity and increased growth. Despite knowing that MC4Rs control food intake, we are yet to understand why defects in the function of the MC4R receptor contribute to rapid linear growth. We show that hyperphagia following germline loss of MC4R in male mice promotes growth while suppressing the growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis. We propose that hyperinsulinaemia promotes growth while suppressing the GH-IGF-1 axis. It is argued that physiological responses essential to maintain energy flux override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. ABSTRACT: Defects in melanocortin-4-receptor (MC4R) signalling result in hyperphagia, obesity and increased growth. Clinical observations suggest that loss of MC4R function may enhance growth hormone (GH)-mediated growth, although this remains untested. Using male mice with germline loss of the MC4R, we assessed pulsatile GH release and insulin-like growth factor-1 (IGF-1) production and/or release relative to pubertal growth. We demonstrate early-onset suppression of GH release in rapidly growing MC4R deficient (MC4RKO) mice, confirming that increased linear growth in MC4RKO mice does not occur in response to enhanced activation of the GH-IGF-1 axis. The progressive suppression of GH release in MC4RKO mice occurred alongside increased adiposity and the progressive worsening of hyperphagia-associated hyperinsulinaemia. We next prevented hyperphagia in MC4RKO mice through restricting calorie intake in these mice to match that of wild-type (WT) littermates. Pair feeding of MC4RKO mice did not prevent increased adiposity, but attenuated hyperinsulinaemia, recovered GH release, and normalized linear growth rate to that seen in pair-fed WT littermate controls. We conclude that the suppression of GH release in MC4RKO mice occurs independently of increased adipose mass, and is a consequence of hyperphagia-associated hyperinsulinaemia. It is proposed that physiological responses essential to maintain energy flux (hyperinsulinaemia and the suppression of GH release) override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Implications of these findings are likely to extend beyond individuals with defects in MC4R signalling, encompassing physiological changes central to mechanisms of growth and energy homeostasis universal to hyperphagia-associated childhood-onset obesity.

Effect of Deletion of Ghrelin-O-Acyltransferase on the Pulsatile Release of Growth Hormone in Mice.

Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat(-/-) mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat(-/-) mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat(-/-) mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat(-/-) mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF-1 release and growth in goat(-/-) mice.

The interplay between metabolic homeostasis and neurodegeneration: insights into the neurometabolic nature of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disease that is characterized by the selective degeneration of upper motor neurons and lower spinal motor neurons, resulting in the progressive paralysis of all voluntary muscles. Approximately 10 % of ALS cases are linked to known genetic mutations, with the remaining 90 % of cases being sporadic. While the primary pathology in ALS is the selective death of upper and lower motor neurons, numerous studies indicate that an imbalance in whole body and/or cellular metabolism influences the rate of progression of disease. This review summarizes current research surrounding the impact of impaired metabolic physiology in ALS. We extend ideas to consider prospects that lie ahead in terms of how metabolic alterations may impact the selective degeneration of neurons in ALS and how targeting of adenosine triphosphate-sensitive potassium (KATP) channels may represent a promising approach for obtaining neuroprotection in ALS.

Altered expression of metabolic proteins and adipokines in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the loss of upper cortical and lower motor neurons. ALS causes death within 2-5years of diagnosis. Diet and body mass index influence the clinical course of disease, however there is limited information about the expression of metabolic proteins and fat-derived cytokines (adipokines) in ALS. In healthy controls and subjects with ALS, we have measured levels of proteins and adipokines that influence metabolism. We find altered levels of active ghrelin, gastric inhibitory peptide (GIP), pancreatic polypeptide (PP), lipocalin-2, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and 8 (IL-8), and tumor necrosis factor alpha (TNFα) in the plasma of ALS patients relative to controls. We also observe a positive correlation between the expression of plasma nerve growth factor (NGF) relative to disease duration, and an inverse correlation between plasma glucagon and the ALS functional rating scale-revised (ALSFRS-R). Further studies are required to determine whether altered expression of metabolic proteins and adipokines contribute to motor neuron vulnerability and how these factors act to modify the course of disease.

Nutrient Sensing Overrides Somatostatin and Growth Hormone-Releasing Hormone to Control Pulsatile Growth Hormone Release.

Pharmacological studies reveal that interactions between hypothalamic inhibitory somatostatin and stimulatory growth hormone-releasing hormone (GHRH) govern pulsatile GH release. However, in vivo analysis of somatostatin and GHRH release into the pituitary portal vasculature and peripheral GH output demonstrates that the withdrawal of somatostatin or the appearance of GHRH into pituitary portal blood does not reliably dictate GH release. Consequently, additional intermediates acting at the level of the hypothalamus and within the anterior pituitary gland are likely to contribute to the release of GH, entraining GH secretory patterns to meet physiological demand. The identification and validation of the actions of such intermediates is particularly important, given that the pattern of GH release defines several of the physiological actions of GH. This review highlights the actions of neuropeptide Y in regulating GH release. It is acknowledged that pulsatile GH release may not occur selectively in response to hypothalamic control of pituitary function. As such, interactions between somatotroph networks, the median eminence and pituitary microvasculature and blood flow, and the emerging role of tanycytes and pericytes as critical regulators of pulsatility are considered. It is argued that collective interactions between the hypothalamus, the median eminence and pituitary vasculature, and structural components within the pituitary gland dictate somatotroph function and thereby pulsatile GH release. These interactions may override hypothalamic somatostatin and GHRH-mediated GH release, and modify pulsatile GH release relative to the peripheral glucose supply, and thereby physiological demand.

Gender differences in autoimmune disease.

Autoimmune diseases are a range of diseases in which the immune response to self-antigens results in damage or dysfunction of tissues. Autoimmune diseases can be systemic or can affect specific organs or body systems. For most autoimmune diseases there is a clear sex difference in prevalence, whereby females are generally more frequently affected than males. In this review, we consider gender differences in systemic and organ-specific autoimmune diseases, and we summarize human data that outlines the prevalence of common autoimmune diseases specific to adult males and females in countries commonly surveyed. We discuss possible mechanisms for sex specific differences including gender differences in immune response and organ vulnerability, reproductive capacity including pregnancy, sex hormones, genetic predisposition, parental inheritance, and epigenetics. Evidence demonstrates that gender has a significant influence on the development of autoimmune disease. Thus, considerations of gender should be at the forefront of all studies that attempt to define mechanisms that underpin autoimmune disease.

Body mass index and dietary intervention: implications for prognosis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult onset, neurodegenerative disease that is characterized by the loss of upper (corticospinal) and lower motor neurons. ALS is a multifactorial disease whereby a combination of genetic and environmental factors may contribute to disease pathogenesis. While the majority of studies indicate that the underlying causes for ALS pathology may be due to multiple defects at the cellular level, factors that have recently been identified to be associated with survival could lead to the development of beneficial interventions. In ALS, a higher pre-morbid body mass index (BMI) and the maintenance of BMI and nutritional state is associated with improved outcome. This review will focus on the associations between body composition and adiposity relative to disease duration and risk, and will discuss current evidence that supports the benefits of improving energy balance, and the maintenance of body mass through nutritional intervention in ALS.

Growth hormone secretion is correlated with neuromuscular innervation rather than motor neuron number in early-symptomatic male amyotrophic lateral sclerosis mice.

GH deficiency is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, therapy with GH and/or IGF-I has not shown benefit. To gain a better understanding of the role of GH secretion in ALS pathogenesis, we assessed endogenous GH secretion in wild-type and hSOD1(G93A) mice throughout the course of ALS disease. Male wild-type and hSOD1(G93A) mice were studied at the presymptomatic, onset, and end stages of disease. To assess the pathological features of disease, we measured motor neuron number and neuromuscular innervation. We report that GH secretion profile varies at different stages of disease progression in hSOD1(G93A) mice; compared with age-matched controls, GH secretion is unchanged prior to the onset of disease symptoms, elevated at the onset of disease symptoms, and reduced at the end stage of disease. In hSOD1(G93A) mice at the onset of disease, GH secretion is positively correlated with the percentage of neuromuscular innervation but not with motor neuron number. Moreover, this occurs in parallel with an elevation in the expression of muscle IGF-I relative to controls. Our data imply that increased GH secretion at symptom onset may be an endogenous endocrine response to increase the local production of muscle IGF-I to stimulate reinnervation of muscle, but that in the latter stages of disease this response no longer occurs.

Development of a methodology for and assessment of pulsatile luteinizing hormone secretion in juvenile and adult male mice.

Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-µL fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events (number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.

Hypothalamic distribution of somatostatin mRNA expressing neurones relative to pubertal and adult changes in pulsatile growth hormone secretion in mice.

The age-associated decline in growth hormone (GH) secretion may be a consequence of the reduction in the number of GH-releasing hormone (GHRH) positive neurones. However, it remains unclear whether an alteration in the number or distribution of somatostatin (SST) neurones contributes to this change. In the present study, we characterised the role of SST in modulating the change in pulsatile GH secretion in male C57Bl/6J mice throughout puberty and into early adulthood. We assessed pulsatile GH secretion in mice at 4, 8 and 16 weeks of age. These ages correspond to early pubertal, early adulthood and adulthood, respectively. We show an elevation in peak, total and pulsatile GH secretion coinciding with periods of rapid linear growth. Using in situ hybridisation and morphometric methods, we mapped the distribution of Sst mRNA expression within the mouse brain relative to this change in pulsatile GH secretion. The results obtained show that altered pulsatile GH secretion in male mice from 4-16 weeks of age does not coincide with a significant change in the number of Sst mRNA expressing neurones or an abundance of Sst mRNA expression throughout the arcuate nucleus (ARC) and periventricular nucleus (PeV). Rather, we observed a progressive decline in Sst mRNA expressing neurones within subnuclei of the paraventricular nucleus at this time. We conclude that structural changes in Sst expression within the PeV and ARC may not reflect the observed decline in pulsatile GH secretion in mice from puberty into early adulthood.

Increased adiposity and insulin correlates with the progressive suppression of pulsatile GH secretion during weight gain.

Pathological changes associated with obesity are thought to contribute to GH deficiency. However, recent observations suggest that impaired GH secretion relative to excess calorie consumption contributes to progressive weight gain and thus may contribute to the development of obesity. To clarify this association between adiposity and GH secretion, we investigated the relationship between pulsatile GH secretion and body weight; epididymal fat mass; and circulating levels of leptin, insulin, non-esterified free fatty acids (NEFAs), and glucose. Data were obtained from male mice maintained on a standard or high-fat diet. We confirm the suppression of pulsatile GH secretion following dietary-induced weight gain. Correlation analyses reveal an inverse relationship between measures of pulsatile GH secretion, body weight, and epididymal fat mass. Moreover, we demonstrate an inverse relationship between measures of pulsatile GH secretion and circulating levels of leptin and insulin. The secretion of GH did not change relative to circulating levels of NEFAs or glucose. We conclude that impaired pulsatile GH secretion in the mouse occurs alongside progressive weight gain and thus precedes the development of obesity. Moreover, data illustrate key interactions between GH secretion and circulating levels of insulin and reflect the potential physiological role of GH in modulation of insulin-induced lipogenesis throughout positive energy balance.

The decline in pulsatile GH secretion throughout early adulthood in mice is exacerbated by dietary-induced weight gain.

The transition between puberty and adulthood is accompanied by a slowing in linear growth. Although GH is a key factor that drives somatic development into adulthood, early adulthood coincides with a reduction in circulating levels of GH. To this extent, a pathological decline in postpubertal GH secretion is detrimental to attainment of peak lean muscle mass and bone mass and promotes adiposity and increases susceptibility to the development of obesity in adulthood. Here we characterized pulsatile GH secretion in C57BL/6J mice at 12 and 16 wk of age. Deconvolution analysis of these measures reveals a reduction in pulsatile GH secretion between 12 and 16 wk of age. Dietary intervention with high-fat feeding at 8 wk of age results in a significant increase in adiposity, the development of glucose intolerance, and hyperinsulinemia. We show the exacerbation of the age-associated decline in pulsatile GH secretion in high-fat-fed mice after 4 wk of dietary intervention (at 12 wk of age), and a further suppression of pulsatile GH secretion by 8 wk of dietary intervention (at 16 wk of age). Suppressed pulsatile secretion of GH did not coincide with an elevation in circulating free fatty acids. Rather, we observed increased hepatic triglyceride content and an eventual decrease in circulating levels of IGF-I. Given the established role of GH in maintaining healthy aging, we anticipate that an advancing of the age-associated decline in pulsatile GH secretion as a consequence of dietary-induced weight gain may have long-term ramifications on adult health.

Impairments to the GH-IGF-I axis in hSOD1G93A mice give insight into possible mechanisms of GH dysregulation in patients with amyotrophic lateral sclerosis.

GH deficiency has been found in subjects with amyotrophic lateral sclerosis (ALS). Disrupted endocrine function could contribute to the progressive muscle loss and hypermetabolism seen in ALS. It is not possible to study all the elements of the GH-IGF-I axis in ALS patients. Consequently, it remains unclear whether dysfunctional GH secretion contributes to disease pathogenesis and why GH and IGF-I directed treatment strategies are ineffective in human ALS. The hSOD1(G93A) transgenic mouse model is useful for the detailed investigation of the pathogenesis of ALS. We report that symptomatic male hSOD1(G93A) transgenic mice exhibit a deficiency in GH secretion similar to that seen in human ALS. Further characterization of the GH-IGF-I axis in hSOD1(G93A) mice reveals central and peripheral abnormalities that are not found in wild-type age-matched controls. Specifically, we observe aberrant endogenous pulsatile GH secretion, reduced pituitary GH content, and decreased circulating levels of IGF-I, indicating global GH deficiency in hSOD1(G93A) mice. Furthermore, a reduction in the expression of the IGF-I receptor α-subunit in skeletal muscle and lumbar spinal cords of hSOD1(G93A) mice suggests impaired IGF-I signaling within these tissues. This is the first account of disrupted GH secretion in a transgenic mouse model of ALS. These observations are essential for the development of effective GH and IGF-I targeted therapies in ALS.

GH does not modulate the early fasting-induced release of free fatty acids in mice.

Fasting results in the mobilization of adipose stores and the elevation of levels of free fatty acids (FFA). In humans, this process is driven by a release in GH. Little is known regarding the role of GH in modulating this process during early stages of fasting in the mouse. Confirmation of the role of GH in modulating FFA release in the fasting mouse is of particular importance given the frequent use of mouse models to study metabolic mechanisms. Here, we correlate the initial release of FFA throughout fasting in mice with pulsatile GH secretion. Observations illustrate the rapid release of FFA in response to food withdrawal. This does not correlate with a rise in GH secretion. Rather, we observed a striking loss in pulsatile secretion of GH throughout the first 6 h of fasting, suggesting that GH does not modulate the initial release of FFA in the mouse in response to fasting. This was confirmed in GH receptor knockout mice, in which we observed a robust fasting-induced rise in FFA. We further illustrate the dynamic relationship between the orexigenic and anorexigenic hormones ghrelin and leptin during fasting in the mouse. Our findings show an initial suppression of leptin and the eventual rise in circulating levels of acyl-ghrelin with fasting. However, altered acyl-ghrelin and leptin secretion occurs well after the rise in FFA and the suppression of GH secretion. Consequently, we conclude that although acyl-ghrelin and leptin may modulate the physiological response to drive food intake, these changes do not contribute to the initial loss of pulsatile GH secretion. Rather, it appears that the suppression of GH secretion in fasting may occur in response to an elevation in fasting levels of FFA or physiological stress. Observations highlight a divergent role for GH in modulating FFA release between man and mouse.

Development of a method for the determination of pulsatile growth hormone secretion in mice.

Measures of pulsatile GH secretion require frequent collection and analysis of blood samples at regular intervals. Due to blood volume constraints, repeat measures of circulating levels of GH in mice remain challenging. Consequently, few observations exist in which the pulsatile pattern of GH secretion in mice have been characterized. To address this, we developed a technique for the collection and analysis of circulating levels of GH at regular and frequent intervals in freely moving mice. This was achieved through the development of a sensitive assay for the detection of GH in small (2 µl) quantities of whole blood. The specificity and accuracy of this assay was validated following guidelines established for single-laboratory validation as specified by the International Union of Pure and Applied Chemistry. We incorporated an established method for tail-clip blood sample collection to determine circulating levels of GH secretion in 36 whole blood samples collected consecutively over a period of 6 h. Resulting measures were characterized by peak secretion periods and interpulse stable baseline secretion periods. Periods characterized by elevated whole blood GH levels consisted of multicomponent peaks. Deconvolution analysis of resulting measures confirmed key parameters associated with pulsatile GH secretion. We show a striking decrease in pulsatile GH secretion in mice after 12-18 h of fasting. This model is necessary to characterize the pulsatile profile of GH secretion in mice and will significantly contribute to current attempts to clarify mechanisms that contribute to the regulation of GH secretion.

Adiponectin regulate growth hormone secretion via adiponectin receptor mediated Ca(2+) signalling in rat somatotrophs in vitro.

Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca(2+) through both the influx of extracellular Ca(2+) and the release of intracellular Ca(2+) stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca(2+) influx through L-type Ca(2+) channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.

Expression of ovarian steroid hormone receptors in tuberoinfundibular dopaminergic neurones during pregnancy and lactation.

During late-pregnancy, tuberoinfundibular dopaminergic (TIDA) neurones, a critical component of the negative-feedback loop regulating prolactin secretion, become unresponsive to the stimulatory effects of prolactin. The change in TIDA responsiveness to prolactin at this time results in a decrease in dopamine secretion and a prolactin surge. As the onset of parturition and the antepartum prolactin surge depend on the withdrawal of progesterone in the presence of oestrogen, it is likely that ovarian steroid hormones mediate this change in TIDA responsiveness. To determine whether ovarian steroids can directly modulate TIDA activity, and whether changes of receptor numbers might contribute to overall steroid-regulation of these neurones, we investigated the level of oestrogen receptor alpha (ERalpha) and progesterone receptor (PR) expression within TIDA neurones during pregnancy and lactation. Animals were sacrificed on dioestrous, days 12, 19 and 21 of pregnancy and day 5 of lactation, and the proportion of TIDA neurones expressing ERalpha or PR, as well as the total number of PR expressing cells within the arcuate nucleus, was determined. Approximately 75% and 55% of tyrosine hydroxylase neurones expressed ERalpha and PR, respectively. Levels of steroid receptor expression within TIDA neurones remained fairly constant, except for an increase in ERalpha on days 12 and 19 of pregnancy compared to dioestrous and lactation day 5. The presence of steroid receptors on TIDA neurones during pregnancy and lactation supports the concept of a direct effect of steroid hormones on these neurones at this time. Thus, steroid hormones may directly act on TIDA neurones to regulate maternal prolactin secretion. The relatively stable level of expression during late pregnancy suggests that a shift in steroid receptor expression during late pregnancy does not contribute to the change in TIDA responsiveness to prolactin at this time.