GDF15 linked to maternal risk of nausea and vomiting during pregnancy.

GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy, including its most severe form, hyperemesis gravidarum (HG), but a full mechanistic understanding is lacking1-4. Here we report that fetal production of GDF15 and maternal sensitivity to it both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally labelled GDF15 variant, we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants, we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with ß-thalassaemia, a condition in which GDF15 levels are chronically high5, report very low levels of nausea and vomiting of pregnancy. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by prepregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Fetally-encoded GDF15 and maternal GDF15 sensitivity are major determinants of nausea and vomiting in human pregnancy.

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

High fat diet causes distinct aberrations in the testicular proteome.

Diet has important effects on normal physiology and the potential deleterious effects of high fat diets and obesity on male reproductive health are being increasingly described. We conducted a histological review of the effects of chronic high fat (HF) diet (using a mouse model fed a 45% fat diet for 21 weeks) with a discovery proteomic study to assess for changes in the abundance of proteins in the testis. Mice on a HF diet became obese and developed glucose intolerance. Using mass spectrometry, we identify 102 proteins affected in the testis of obese mice. These included structural proteins important for the blood testis barrier (filamin A, FLNA), proteins involved in oxidative stress responses (spermatogenesis associated 20, SPATA-20) and lipid homoeostasis (sterol regulatory element-binding protein 2, SREBP2 and apolipoprotein A1, APOA1). In addition, an important regulator protein paraspeckle component 1, PSPC-1, which interacts with the androgen receptor was significantly downregulated. Proteomic data was validated using both Western blotting and immunostaining which confirmed and localised protein expression in both mouse and human testis using biopsy specimens. This study focused mainly on the abnormalities that occurred at the protein level and as a result, we have identified several candidate proteins and conducted pathway analysis around the effects of HF diet on the testis providing novel insights not previously described. Some of the identified targets could be targeted therapeutically and future work is directed in this area.

Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice.

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.

The hypoxia response pathway and ß-cell function.

ß-cells sense glucose and secrete appropriate amounts of insulin by coupling glucose uptake and glycolysis with quantitative ATP production via mitochondrial oxidative pathways. Therefore, oxidative phosphorylation is essential for normal ß-cell function. Multiple cell types adapt to hypoxia by inducing a transcriptional programme coordinated by the transcription factor hypoxia-inducible factor (HIF). HIF activity is regulated by the von Hippel-Lindau (Vhl) protein, which targets the HIFα subunit for proteasomal degradation in the presence of oxygen. Several recent studies have shown that Vhl deletion in ß-cells results in Hif1α activation, impaired glucose-stimulated insulin secretion (GSIS) and glucose intolerance. This was found to be because of alterations in ß-cell gene expression inducing a switch from aerobic glucose metabolism to anaerobic glycolysis, thus disrupting the GSIS triggering pathway. Situations in which islets may become hypoxic are discussed, in particular islet transplantation which has been reported to cause islet hypoxia because of an inadequate blood supply post-transplant. Aside from this principal role for HIF in negatively regulating ß-cell glucose sensing, other aspects of hypoxia signalling are discussed including ß-cell differentiation, development and vascularization. In conclusion, recent studies clearly show that hypoxia response mechanisms can negatively impact on glucose sensing mechanisms in the ß-cell and this has the potential to impair ß-cell function in a number of physiological and clinical situations.

5-HT inhibition of rat insulin 2 promoter Cre recombinase transgene and proopiomelanocortin neuron excitability in the mouse arcuate nucleus.

A number of anti-obesity agents have been developed that enhance hypothalamic 5-HT transmission. Various studies have demonstrated that arcuate neurons, which express proopiomelanocortin peptides (POMC neurons), and neuropeptide Y with agouti-related protein (NPY/AgRP) neurons, are components of the hypothalamic circuits responsible for energy homeostasis. An additional arcuate neuron population, rat insulin 2 promoter Cre recombinase transgene (RIPCre) neurons, has recently been implicated in hypothalamic melanocortin circuits involved in energy balance. It is currently unclear how 5-HT modifies neuron excitability in these local arcuate neuronal circuits. We show that 5-HT alters the excitability of the majority of mouse arcuate RIPCre neurons, by either hyperpolarization and inhibition or depolarization and excitation. RIPCre neurons sensitive to 5-HT, predominantly exhibit hyperpolarization and pharmacological studies indicate that inhibition of neuronal firing is likely to be through 5-HT(1F) receptors increasing current through a voltage-dependent potassium conductance. Indeed, 5-HT(1F) receptor immunoreactivity co-localizes with RIPCre green fluorescent protein expression. A minority population of POMC neurons also respond to 5-HT by hyperpolarization, and this appears to be mediated by the same receptor-channel mechanism. As neither POMC nor RIPCre neuronal populations display a common electrical response to 5-HT, this may indicate that sub-divisions of POMC and RIPCre neurons exist, perhaps serving different outputs.

Pancreatic deletion of insulin receptor substrate 2 reduces beta and alpha cell mass and impairs glucose homeostasis in mice.

AIMS/HYPOTHESIS: Insulin signalling pathways regulate pancreatic beta cell function. Conditional gene targeting using the Cre/loxP system has demonstrated that mice lacking insulin receptor substrate 2 (IRS2) in the beta cell have reduced beta cell mass. However, these studies have been complicated by hypothalamic deletion when the RIPCre (B6.Cg-tg(Ins2-cre)25Mgn/J) transgenic mouse (expressing Cre recombinase under the control of the rat insulin II promoter) is used to delete floxed alleles in insulin-expressing cells. These features have led to marked insulin resistance making the beta cell-autonomous role of IRS2 difficult to determine. To establish the effect of deleting Irs2 only in the pancreas, we generated PIrs2KO mice in which Cre recombinase expression was driven by the promoter of the pancreatic and duodenal homeobox factor 1 (Pdx1, also known as Ipf1) gene. MATERIALS AND METHODS: In vivo glucose homeostasis was examined in PIrs2KO mice using glucose tolerance and glucose-stimulated insulin secretion tests. Endocrine cell mass was determined by morphometric analysis. Islet function was examined in static cultures and by performing calcium imaging in Fluo3am-loaded beta cells. Islet gene expression was determined by RT-PCR. RESULTS: The PIrs2KO mice displayed glucose intolerance and impaired glucose-stimulated insulin secretion in vivo. Pancreatic insulin and glucagon content and beta and alpha cell mass were reduced. Glucose-stimulated insulin secretion and calcium mobilisation were attenuated in PIrs2KO islets. Expression of the Glut2 gene (also known as Slc2a2) was also reduced in PIrs2KO mice. CONCLUSIONS/INTERPRETATION: These studies suggest that IRS2-dependent signalling in pancreatic islets is required not only for the maintenance of normal beta and alpha cell mass but is also involved in the regulation of insulin secretion.

Liver-specific deletion of insulin receptor substrate 2 does not impair hepatic glucose and lipid metabolism in mice.

AIMS/HYPOTHESIS: Hepatic insulin resistance is thought to be a critical component in the pathogenesis of type 2 diabetes but the role of intrinsic insulin signalling pathways in the regulation of hepatic metabolism remains controversial. Global gene targeting in mice and in vitro studies have suggested that IRS2 mediates the physiological effects of insulin in the liver. Reduced hepatic production of IRS2 is found in many cases of insulin resistance. To investigate the role of IRS2 in regulating liver function in vivo, we generated mice that specifically lack Irs2 in the liver (LivIrs2KO). MATERIALS AND METHODS: Hepatic insulin signalling events were examined in LivIrs2KO mice by western blotting. Glucose homeostasis and insulin sensitivity were assessed by glucose tolerance tests and hyperinsulinaemic-euglycaemic clamp studies. The effects of high-fat feeding upon glucose homeostasis were also determined. Liver function tests were performed and expression of key metabolic genes in the liver was determined by RT-PCR. RESULTS: Proximal insulin signalling events and forkhead box O1 and A2 function were normal in the liver of LivIrs2KO mice, which displayed minimal abnormalities in glucose and lipid homeostasis, hepatic gene expression and liver function. In addition, hepatic lipid homeostasis and the metabolic response to a high-fat diet did not differ between LivIrs2KO and control mice. CONCLUSIONS/INTERPRETATION: Our findings suggest that liver IRS2 signalling, surprisingly, is not required for the long-term maintenance of glucose and lipid homeostasis, and that extra-hepatic IRS2-dependent mechanisms are involved in the regulation of these processes.

Adrenomedullin: receptor and signal transduction.

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.

Insulin receptor substrate proteins and neuroendocrine function.

A family of insulin receptor substrate (IRS) proteins mediates the pleiotropic effects of insulin and insulin-like growth factor 1 (IGF-1) on cellular function by recruiting several intracellular signalling networks. Conventional murine knockout strategies have started to reveal distinct physiological roles for the IRS proteins. Deletion of Irs1 produces a mild metabolic phenotype with compensated insulin resistance but also causes marked growth retardation. In contrast, mice lacking IRS-2 display nearly normal growth but develop diabetes owing to a combination of peripheral insulin resistance and beta-cell failure. As well as the classical metabolic events regulated by insulin signalling pathways, studies in lower organisms have implicated insulin/IGF-1 signalling pathways in the control of food intake and reproductive function. Our analysis of IRS-2 knock-out mice shows that female mice are infertile owing to defects in the hypothalamus, pituitary and gonad. IRS-2(-/-) mice have small, anovulatory ovaries with reduced numbers of follicles. Levels of the pituitary hormones luteinizing hormone and prolactin and gonadal steroids are low in these animals. Pituitaries of IRS-2(-/-) animals are decreased in size and contain reduced numbers of gonadotrophs. Additionally, IRS-2(-/-) females display increased food intake and develop obesity, despite elevated leptin levels, suggesting abnormalities in hypothalamic function. Coupled with recent observations that brain-specific deletion of the insulin receptor causes a similar phenotype, these findings implicate IRS signalling pathways in the neuroendocrine regulation of reproduction and energy homeostasis.

IRS-2 pathways integrate female reproduction and energy homeostasis.

Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.

Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo.

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.

Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation.

Growth factor induced activation of phosphoinositide 3-kinase and protein kinase B (PKB) leads to increased activity of the mammalian target of rapamycin (mTOR). This subsequently leads to increased phosphorylation of eIF4E binding protein-1 (4EBP1) and activation of p70 ribosomal S6 protein kinase (p70(S6K)), both of which are important steps in the stimulation of protein translation. The stimulation of translation is attenuated in cells deprived of amino acids and this is associated with the attenuation of 4EBP1 phosphorylation and p70(S6K) activation. It has been suggested that PKB regulates mTOR function by phosphorylation although direct phosphorylation of mTOR by PKB has not been demonstrated previously. In the present work, we have found that PKB directly phosphorylates mTOR and, using phosphospecific antibodies, we have shown this phosphorylation occurs at Ser(2448). Insulin also induces phosphorylation on Ser(2448) and this effect is blocked by wortmannin but not rapamycin, consistent with the effect being mediated by PKB. Amino-acid starvation rapidly attenuated the reactivity of the Ser(2448) phosphospecific antibody with mTOR and this could not be restored by either insulin stimulation of cells or incubation with PKB in vitro. Our findings demonstrate that mTOR is a direct target for PKB and support the conclusion that regulation of phosphorylation of Ser(2448) is a point of convergence for the counteracting regulatory effects of growth factors and amino acid levels.

Irs-2 coordinates Igf-1 receptor-mediated beta-cell development and peripheral insulin signalling.

Insulin receptor substrates (Irs proteins) mediate the pleiotropic effects of insulin and Igf-1 (insulin-like growth factor-1), including regulation of glucose homeostasis and cell growth and survival. We intercrossed mice heterozygous for two null alleles (Irs1+/- and Irs2+/-) and investigated growth and glucose metabolism in mice with viable genotypes. Our experiments revealed that Irs-1 and Irs-2 are critical for embryonic and post-natal growth, with Irs-1 having the predominant role. By contrast, both Irs-1 and Irs-2 function in peripheral carbohydrate metabolism, but Irs-2 has the major role in beta-cell development and compensation for peripheral insulin resistance. To establish a role for the Igf-1 receptor in beta-cells, we intercrossed mice heterozygous for null alleles of Igf1r and Irs2. Our results reveal that Igf-1 receptors promote beta-cell development and survival through the Irs-2 signalling pathway. Thus, Irs-2 integrates the effects of insulin in peripheral target tissues with Igf-1 in pancreatic beta-cells to maintain glucose homeostasis.

Insulin receptor substrate-2 is not necessary for insulin- and exercise-stimulated glucose transport in skeletal muscle.

Insulin receptor substrate-2-deficient (IRS2(-/-)) mice develop type 2 diabetes. The purpose of this study was to determine whether there is a defect in basal, insulin-, and exercise-stimulated glucose transport in the skeletal muscle of these animals. IRS2(-/-) and wild-type (WT) mice (male, 8-10 weeks) exercised on a treadmill for 1 h or remained sedentary. 2-Deoxyglucose (2DG) uptake was measured in isolated soleus muscles incubated in vitro in the presence or absence of insulin. Resting blood glucose concentration in IRS2(-/-) mice (10.3 mM) was higher than WT animals (4.1 mM), but there was a wide range among the IRS2(-/-) mice (3-25 mM). Therefore, IRS2(-/-) mice were divided into two subgroups based on blood glucose concentrations (IRS2(-/-)L < 7.2 mM, IRS2(-/-)H > 7.2 mM). Only IRS2(-/-)H had lower basal, exercise-, and submaximally insulin-stimulated 2DG uptake, while maximal insulin-stimulated 2DG uptake was similar among the three groups. The ED(50) for insulin to stimulate 2DG uptake above basal in IRS2(-/-)H was higher than WT and IRS2(-/-)L mice, suggesting insulin resistance in the skeletal muscle from the IRS2(-/-) mice with high blood glucose concentrations. Furthermore, resting blood glucose concentrations from all groups were negatively correlated to submaximally insulin-stimulated 2DG uptake (r(2) = 0.33, p < 0.01). Muscle GLUT4 content was significantly lower in IRS2(-/-)H mice compared with WT and IRS2(-/-)L mice. These results demonstrate that the IRS2 protein in muscle is not necessary for insulin- or exercise-stimulated glucose transport, suggesting that the onset of diabetes in the IRS2(-/-) mice is not due to a defect in skeletal muscle glucose transport; hyperglycemia may cause insulin resistance in the muscle of IRS2(-/-) mice.

Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses.

Disruption of IRS-2 causes type 2 diabetes in mice.

Human type 2 diabetes is characterized by defects in both insulin action and insulin secretion. It has been difficult to identify a single molecular abnormality underlying these features. Insulin-receptor substrates (IRS proteins) may be involved in type 2 diabetes: they mediate pleiotropic signals initiated by receptors for insulin and other cytokines. Disruption of IRS-1 in mice retards growth, but diabetes does not develop because insulin secretion increases to compensate for the mild resistance to insulin. Here we show that disruption of IRS-2 impairs both peripheral insulin signalling and pancreatic beta-cell function. IRS-2-deficient mice show progressive deterioration of glucose homeostasis because of insulin resistance in the liver and skeletal muscle and a lack of beta-cell compensation for this insulin resistance. Our results indicate that dysfunction of IRS-2 may contribute to the pathophysiology of human type 2 diabetes.

Signalling pathways involved in the mitogenic effects of cAMP.

1. Elevation of intracellular cyclic AMP (cAMP) is a potent mitogenic signal for a number of cell types, including Swiss 3T3 cells, thyroid epithelial cells and the somatotroph cells of the anterior pituitary. 2. Activation of the mitogen-activated protein kinase (MAPK) cascade has been shown to underlie the mitogenic effects of many growth factors. However, the precise relationship between the mitogenic effects of cAMP and the MAPK cascade is not fully defined. 3. In Swiss 3T3 cells, elevation of cAMP did not stimulate kinases at all three levels of the MAPK cascade. Additionally, blockade of the MAPK pathway failed to inhibit cAMP-stimulated DNA synthesis. 4. Mitogenic combinations of cAMP strongly stimulated the phosphorylation and activation of the serine/threonine kinase p70 S6 kinase, p70S6K, an effect that was inhibited by rapamycin. This agent markedly inhibited cAMP-stimulated DNA synthesis, suggesting a critical role for p70S6K in cAMP mitogenic signalling. 5. Thus, multiple parallel but distinct signalling pathways may be involved in the action of mitogens. This redundancy has important implications for the pathogenesis and treatment of conditions characterized by inappropriate activation of growth factor signalling pathways.

Regulation of anterior pituitary galanin and vasoactive intestinal peptide by oestrogen and prolactin status.

The neuropeptides vasoactive intestinal peptide (VIP) and galanin are synthesized in the anterior pituitary, galanin in the lactotroph and VIP probably in another cell type, and both stimulate prolactin secretion. Oestrogen regulates anterior pituitary VIP and galanin, galanin expression reflecting physiological variation in oestrogen status, whilst VIP is induced by pharmacological concentrations of oestrogen. Implanting anterior pituitaries under the renal capsule to induce hyperprolactinaemia we studied the regulation of anterior pituitary VIP and galanin synthesis and storage by prolactin and its interaction with oestrogen status. Five groups of animals were studied: control, hypophysectomized implanted, implanted, hyperoestrogenized (oestradiol-17 beta; 250 micrograms/day) and hyperoestrogenized implanted. Spontaneously cycling animals were followed through two cycles prior to implanting and were maintained for at least 1 week and then killed once they were in dioestrus. Circulating prolactin levels were significantly elevated in implanted animals but not in hypophysectomized implanted animals compared with controls. There was a more marked increase in prolactin levels in hyperoestrogenized animals and hyperoestrogenized implanted animals, with no significant difference between these two groups. Native anterior pituitary galanin and VIP content was suppressed in implanted animals, and markedly increased in hyperoestrogenized animals. Pituitary implantation only marginally reduced the effect of hyperoestrogenization on galanin content but abolished the effect of hyperoestrogenization on VIP content. Implant peptide content was suppressed to less than 10% of native anterior pituitary content. Galanin was not detected in implants from hypophysectomized-implanted animals but implant VIP content was unaffected by hypophysectomy. VIP content was increased in implants from hyperoestrogenized implanted animals but implant galanin content was unaffected by hyperoestrogenization. Peptide mRNA levels changed in parallel with peptide content except that the implant galanin mRNA levels were increased by hyperoestrogenization. Thus it appears that prolactin negatively regulates anterior pituitary galanin and VIP gene expression and content, probably due to a direct effect on the anterior pituitary and by altered secretion of hypothalamic factors. Oestrogen is a potent stimulus to expression of both peptide genes. Its positive effect on anterior pituitary peptide gene expression and content is greatly diminished by the effect of implant-induced hyperprolactinaemia, suggesting that circulating prolactin levels may be controlled by a negative feedback effect of prolactin on galanin and VIP. A similar effect of hyperoestrogenization is observed in the implants, except that galanin content remains at a low level, suggesting that the combination of hyperoestrogenization and the absence of dopamine may lead to uncontrolled release of high levels of galanin.