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.

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.

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.

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.

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.

Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice.

Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism.

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.

Evidence for neuromedin-B synthesis in the rat anterior pituitary gland.

Neuromedin-B shows a widespread distribution throughout mammalian neural and peripheral tissues and may be involved in the modulation of a variety of physiological processes. The highest concentrations of neuromedin-B are found in the anterior pituitary gland, suggesting that it may be of physiological importance within this tissue. To examine this hypothesis, we have used Northern blotting to demonstrate that neuromedin-B is present in the anterior pituitary as a result of local synthesis and have examined the effects of endocrine manipulations on its mRNA and immunoreactive peptide content. In thyroidectomized male rats, neuromedin-B content was decreased (104.1 +/- 5.8 vs. control, 390.9 +/- 23.3 fmol/gland; P less than 0.01) as was its mRNA (7 +/- 1.3% vs. 100%; P less than 0.014), while treatment of intact animals with T4 produced no effect on either peptide content or mRNA. Adrenalectomized male rats showed a significant increase in both neuromedin-B content (313.8 +/- 11.8 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.05) and mRNA (377 +/- 27% vs. 100%; P less than 0.014), while dexamethasone treatment increased peptide content (347.8 +/- 32 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.01) without any effect on mRNA levels. In female rats, ovariectomy decreased neuromedin-B content (132.4 +/- 13.3 vs. control, 335.0 +/- 37.2 fmol/gland; P less than 0.01) and mRNA levels (6 +/- 2% vs. 100%; P less than 0.014) while estrogen treatment of both ovariectomized and intact rats produced large increases in neuromedin-B (887.8 +/- 114.4 and 1328 +/- 175 fmol/gland, respectively, vs. control, 335.0 +/- 37.2 fmol/gland; both P less than 0.01) and its mRNA (246 +/- 18% and 378 +/- 31%, respectively, vs. 100%; both P less than 0.014). In castrated male rats, no significant alteration in peptide content was observed, and treatment of both castrated and intact male rats with testosterone was similarly without effect. These results demonstrate that 1) neuromedin-B is locally synthesized within the rat anterior pituitary gland; and 2) the local production of neuromedin-B is influenced by endocrine status, which is consistent with an autocrine/paracrine role for this peptide in this tissue.

Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells.

Our results demonstrate that the novel vasoactive regulatory peptide adrenomedullin is a potent mitogen for Swiss 3T3 cells. Acting via a specific adrenomedullin receptor, it stimulates a dose-dependent increase in DNA synthesis in synergy with insulin. Additionally, adrenomedullin stimulates further progression through the cell cycle resulting in cell proliferation, an effect that was further enhanced by the presence of insulin. Adrenomedullin rapidly induces accumulation of intracellular cAMP but does not stimulate an increase in intracellular Ca2+, activation of protein kinase C, or tyrosine phosphorylation of intracellular substrates. Adrenomedullin-stimulated mitogenesis is markedly enhanced in Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha, which are highly sensitive to agents that elevate cAMP, and is inhibited by the PKA inhibitor H-89. Adrenomedullin is, thus, identified as a novel mitogenic regulatory peptide acting via cAMP.

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.

Technical note: inspiration or expiration? Reducing motion artefact in digital subtraction arch angiography of the extracranial carotid arteries.

100 consecutive patients underwent digital subtraction aortic arch angiography for suspected carotid bifurcation disease. Patients were prospectively randomized to suspend respiration in inspiration or expiration during image acquisition. Images were assessed qualitatively as "excellent", "adequate" or "poor". 92 studies were available for review. Adequate and poor groups were combined into a non-excellent group due to the small numbers that were scored poor. There were 27 (53%) excellent, 24 (47%) non-excellent in the expiration group and 12 (29%) excellent, 29 (71%) non-excellent in the inspiration group. Analysis of angiogram scores gave chi 2(1) = 4.3; p = 0.038, indicating that the higher angiogram scores of the expiration group were statistically significant. Pixel shifting was used in 61 cases (66.3%). The proportion was 34.4% lower in the expiration group (95% CI; 17-52%) with chi 2(1) = 10.5; p = 0.0012 indicating that the reduction in the use of pixel shifting for the expiration group was statistically significant. The superiority of arch angiograms obtained with the patient suspending respiration in expiration rather than inspiration has been demonstrated. This was achieved with significantly reduced use of pixel shifting, encouraging greater confidence in the validity of the angiographic findings. The technique is free of cost or time implications.

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.

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.

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.

Dissociation of cAMP-stimulated mitogenesis from activation of the mitogen-activated protein kinase cascade in Swiss 3T3 cells.

Elevation of intracellular cAMP by forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and prostaglandin E1, in synergy with insulin, stimulated DNA synthesis in quiescent Swiss 3T3 cells to the same level achieved by platelet-derived growth factor (PDGF) or bombesin. Both forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate stimulated a significant increase in cell number which, in the presence of insulin, reached the same levels achieved with PDGF. Treatment with either PDGF or bombesin caused a marked and persistent stimulation of p42MAPK and p44MAPK. In striking contrast, no activation was seen with mitogenic combinations of cAMP as shown by three different assays. Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha subunit were 100-fold more sensitive to the mitogenic effects of forskolin but in this distinct cellular model forskolin did not activate p42MAPK. Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a 60% decrease in PDGF-stimulated p42 MAPK activation, but there was no inhibition of the mitogenic effect of forskolin in these cells. Furthermore, the upstream kinases MEK-1/MEK-2 and p74raf-1 were not activated by mitogenic combinations of cAMP while PDGF caused marked stimulation of their activity. Treatment of 3T3 cells with forskolin attenuated PDGF-stimulated p74raf-1 and p42MAPK activation but enhanced the mitogenic effects of this agent. Mitogenic combinations of cAMP strongly stimulated the phosphorylation and activation of 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 signaling. These results demonstrate that cAMP-induced mitogenesis can be dissociated from activation of the mitogen-activated protein kinase cascade and that this is not an obligatory point of convergence in mitogenic signaling in Swiss 3T3 cells.

Reduced requirement of mitogen-activated protein kinase (MAPK) activity for entry into the S phase of the cell cycle in Swiss 3T3 fibroblasts stimulated by bombesin and insulin.

Bombesin induced a marked and persistent activation of the mitogen-activated protein kinase kinase-1 (MEK-1), p42(mapk) and p90(rsk) in Swiss 3T3 cells by a pathway that was independent of p74(raf-1) but dependent on the activity of protein kinase C. Pretreatment of the cells with a specific inhibitor of MEK-1, PD 098059, markedly reduced the early and abolished the sustained phase of bombesin-induced p42(mapk) activation. In addition, PD 098059 prevented bombesin-induced DNA synthesis and progression of the cells through the cell cycle, indicating that the mitogenic effect of bombesin is dependent on the activation of p42(mapk). However, in the presence of insulin, which neither stimulated p42(mapk) activation nor DNA synthesis on its own in Swiss 3T3 cells, bombesin potently stimulated DNA synthesis even at concentrations of PD 098059 (15 microM) that completely abolished the mitogenic effect of bombesin alone. Furthermore, Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a marked decrease in the mitogenic effect of bombesin. In contrast, the combination of bombesin and insulin strongly stimulated DNA synthesis in these cells to levels comparable with that obtained in the wild type cells. Thus, our data demonstrate that insulin dramatically reduced the requirement for the mitogen-activated protein kinase pathway for reinitiation of DNA synthesis in bombesin-treated Swiss 3T3 cells and consequently indicate that the contribution of the mitogen-activated protein kinase cascade to mitogenesis depends on the combination of extracellular signals that are used to stimulate these cells.