Receptor regulation of phosphoinositide 3-hydroxykinase in the NG115-401L-C3 neuronal cell line: stimulation by insulin-like growth factor-I

The activation of phosphoinositide 3-hydroxykinase (P13K) is currently believed to represent the critical regulatory event which leads to the production of a novel intracellular signal. We have examined the control of this pathway by a number of cell-surface receptors in NG115-401L-C3 neuronal cells. Insulin-like growth factor-I stimulated the accumulation of 3-phosphorylated inositol lipids in intact cells and the appearance of P13K in antiphosphotyrosine-antibody-directed immunoprecipitates prepared from lysed cells, suggesting that P13K had been activated by a mechanism involving a protein tyrosine kinase. In contrast, P13K in these cells was not regulated by a variety of G-protein-coupled receptors, nerve growth factor acting via a low affinity receptor, or receptors for transforming growth factor-beta and interleukin-1. The receptor-specificity of P13K activation in these cells places significant constraints on the possible physiological function(s) of this pathway.

Role of the dsRNA-dependent protein kinase (PKR) in the attenuation of protein loss from muscle by insulin and insulin-like growth factor-I (IGF-I)

Proteolysis-inducing factor (PIF), a tumour-produced cachectic factor, induced a dose-dependent decrease in protein synthesis in murine myotubes, together with an increase in phosphorylation of eucaryotic initiation factor 2 (eIF2) on the alpha-subunit. Both insulin (1 nM) and insulin-like growth factor I (IGF-I) (13.2 nM) attenuated the depression of protein synthesis by PIF and the increased phosphorylation of eIF2alpha, by inhibiting the activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) by induction of protein phosphatase 1. A low-molecular weight inhibitor of PKR also reversed the depression of protein synthesis by PIF to the same extent, as did insulin and IGF-I. Both insulin and IGF-I-stimulated protein synthesis in the presence of PIF, and this was attenuated by Salubrinal, an inhibitor of phospho eIF2alpha phosphatase, suggesting that at least part of this action was due to their ability to inhibit phosphorylation of eIF2alpha. Both insulin and IGF-I also attenuated the induction of protein degradation in myotubes induced by PIF, this effect was also attenuated by Salubrinal. These results suggest an alternative mechanism involving PKR to explain the effect of insulin and IGF-I on protein synthesis and degradation in skeletal muscle in the presence of catabolic factors.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.

The role of insulin and the insulin-like growth factors in the proliferation of the rat thymic lymphocyte

Concanavalin A, provoked a 35-fold increase in the rate of proliferation of rat thymocytes. Insulin (10-6M), and insulin-like growth factor I (10-10M) approximately doubled the rate of DNA synthesis. Both of these structurally related molecules acted through the type I insulin-like growth factor receptor. The sequential addition of Concanavalin A and insulin, promoted a much greater proliferative response than to either of the two agonists alone. Insulin also increased the uptake of glucose and amino acids by the cells. Glucose uptake was enhanced at insulin concentrations of 10-6M and 10-10M. Amino acid uptake was more strongly affected at the higher concentration. Insulin-like growth factor I (10-11M) also enhanced amino acid uptake. The effects of insulin on metabolism were mediated by both insulin and type I insulin-like growth factor receptors. These effects were greatly enhanced after a pre-treatment with Concanavalin A. Concanavalin A provided a primary mitogenic signal to the cells. Amongst the responses was an increased expression of insulin and/or type I insulin-like growth factor receptors. The consequent enhanced cellular sensitivity to these agonists, enabled them to facilitate the passage of the cells through the cell cycle by: i) providing a secondary mitogenic signal, and ii) promoting the uptake of raw materials and energy substrates. The initiation of DNA synthesis and passage through the cell cycle was thus punctuated by the sequential expression of various cell surface receptors. This regulated cellular sensitivity, enabling them to react in a precisely orchestrated fashion to hormones and other molecules in their environment. The intracellular mechanism of insulin action remains an enigma. Although the presence of extracellular calcium was essential for insulin stimulation of amino acid uptake and DNA synthesis, the cation did not subserve a direct mediator function. Insulin promoted an increase in intracellular pH, which was mediated by the Na+/H+ antiport. Other mechanisms were probably also involved in mediating the full cellular response to insulin.

Maternal periconceptional and gestational low protein diet affects mouse offspring growth, cardiovascular and adipose phenotype at 1 year of age

Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (Insr) and insulin-like growth factor I receptor (Igf1r) in retroperitoneal fat was significantly elevated in Emb-LPD females (P&0.05), whilst Emb-LPD males displayed significantly decreased expression of the mitochondrial uncoupling protein 1 (Ucp1) gene compared to NPD offspring. LPD females displayed significantly increased expression of Ucp1 in interscapular brown adipose tissue when compared to NPD offspring. Our results demonstrate that aging offspring body weight, cardiovascular and adiposity homeostasis can be programmed by maternal periconceptional nutrition. These adverse outcomes further exemplify the criticality of dietary behaviour around the time of conception on long-term offspring health. © 2011 Watkins et al.

Changes in inositol lipids and phosphates after stimulation of the MAS-transfected NG115-401L-C3 cell line by mitogenic and non-mitogenic stimuli

A neuronal cell line (NG115-401L-C3) was stimulated by mitogenic (angiotensin) and non-mitogenic (bradykinin) peptides and examined for the time course of changes in the levels of radiolabelled inositol phosphates and phospholipids. Both peptides stimulated the time-dependent production of Ins(1,4,5)P3 and related metabolites. Bradykinin caused a much larger increase in Ins(1,4,5)P3 than did angiotensin. However, both peptides stimulated similar rises in the levels of Ins(1,3,4)P3 and InsP4. Bradykinin but not angiotensin, caused a rapid (within 2 s) fall in the levels of PtdIns(4,5)P2 and PtdIns(4)P. Serum pretreatment of the cells caused a 2-3-fold potentiation of both the responses to bradykinin and angiotensin. Although significant levels of PtdIns(3)P were detected in resting cells neither mitogenic (angiotensin, insulin-like growth factor I, transforming growth factor beta) nor non-mitogenic (bradykinin, nerve growth factor interleukin-1) receptor activation changed its levels, arguing against regulation of either PtdIns 3-kinase or PtdIns(3)P phosphatase. We conclude that, as judged by the levels of its product. PtdIns(3)P, the enzyme PtdIns 3-kinase is not activated. This questions the significance of this activity in the receptor-mediated initiation of DNA synthesis.

Angiotensin II directly inhibits protein synthesis in murine myotubes

Angiotensin II (Ang II) has been implicated in muscle protein loss in cachexia. To determine whether the Ang I/II system directly inhibits protein synthesis in muscle their effect has been monitored in vitro using murine myotubes as a surrogate model system. Ang I inhibited protein synthesis by 40-50% over the concentration range of 0.05-2.5 μM within 30 min of addition, and the inhibition remained relatively constant over 24 h. The effect was attenuated by co-incubation with the angiotensin converting enzyme inhibitor imidaprilat (50 μM) suggesting that inhibition of protein synthesis was due to the formation of Ang II. Ang II also inhibited protein synthesis by 40-50% over the concentration range of 0.1-5 μM, and the inhibition also remained relatively constant between 30 min and 24 h after addition. The effect was attenuated by insulin-like growth factor-1 (IGF-1) (25-100 ng/ml). Thus, Ang I/II have the ability to induce muscle atrophy through inhibition of protein synthesis. © 2005 Elsevier Ireland Ltd. All rights reserved.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Receptor for activated C Kinase 1 protein binds to and activates the human estrogen receptor α

The classical concept of estrogen receptor (ER) activation is that steroid passes the cell membrane, binds to its specific protein receptor in the cell's cytoplasm and the steroid-receptor complex travels to the nucleus where it activates responsive genes. This basic idea has been challenged by results of experiments demonstrating insulin-like growth factor 1 (IGF-1) activation of the ER in the complete absence of estrogen suggesting at least one other mechanism of ER activation not involving steroid. One explanation is that activation of the cell surface IGF-1 receptor leads to synthesis of an intracellular protein(s) able to bind to and stimulate the ER. Based on results using the two-hybrid system, coimmunoprecipitation and transfection-luciferase assays, we herein show that one of these proteins could well be receptor for activated C kinase 1 (RACK-1). Using the human ER type α (ER-α) as bait, a cloned complementary deoxyribonucleic acid (cDNA) library from IGF-1 treated human breast cancer MCF-7 cells was screened for ER-α - protein interactions. Many positive clones were obtained which contained the RACK-1 cDNA sequence. Coimmunoprecipitation of in-vitro translation products of the ER-α and RACK-1 confirmed the interaction between the two proteins. Transfection studies using the estrogen response element spliced to a luciferase reporter gene revealed that constitutive RACK-1 expression was able to powerfully stimulate ER-α activity under estrogen-free conditions. This effect could be enhanced by 17β-estradiol (E2) and blocked by tamoxifen, an E2 antagonist. These results show that RACK-1 is able to activate the ER-α in the absence of E2, although together with the latter, enhanced effects occur. Since RACK-1 gene expression is stimulated by IGF-1, it is distinctly possible that RACK-1 is the mediator of the stimulatory effects of IGF-1 on ER-α. © 2014 JMS.

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in placenta growth factor (PlGF) deficiency

Placenta growth factor (PlGF) deficient mice are fertile at a Mendelian ratio. Interestingly, low maternal plasma levels of PlGF are strongly associated with early onset of preeclampsia, a pregnancy hypertensive disorder characterised by high blood pressure, proteinuria and fetal growth restriction. PlGF is increasingly being recognised as an early diagnostic biomarker, but the physiological importance of PlGF in the pathogenesis of preeclampsia is unknown. We investigated whether the decreased levels of PlGF in pregnancy exacerbate the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1 and the potential of hydrogen sulphide to ameliorate these effects. Pregnant PlGF−/− mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at 1 × 109 pfu/ml at E10.5 and mean arterial blood pressure (MAP), biochemical and histological analysis of maternal kidney, placenta and embryos were assessed at the end of pregnancy. Ad-sFlt-1 significantly increased MAP and induced severe glomerular endotheliosis in PlGF−/− mice compared to wild-type animals. Soluble Flt-1 also significantly elevated albumin–creatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury. Furthermore, sFlt-1 over expression increased fetal resorption rate in the PlGF−/− mice and promoted abnormal placental vascularisation. To determine whether placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/− placentas and embryos in dams and exposed to high sFlt-1 environment. These mothers showed reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF−/− mice. Furthermore, treatment with hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria observed in Ad-sFlt-1 treated pregnant PlGF−/− mice. Our study shows that placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 associated with preeclampsia and activation of the hydrogen sulphide pathway may rescue preeclampsia phenotypes even under low PlGF environment.

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in Placenta Growth Factor (PlGF) deficiency

INTRODUCTION: Low circulating levels of placenta growth factor (PlGF) is strongly associated with the onset of preeclampsia, a maternal hypertensive disorder characterized by high blood pressure and proteinuria after 20 weeks of gestation. Although, PlGF-deficient mice are born healthy and fertile at a Mendelian ratio, the physiological importance of PlGF in the pathogenesis of preeclampsia is unclear. We hypothesised that decreased levels of PlGF in pregnancy exacerbates the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1. METHODS: Pregnant PlGF-/- mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at high (i) 1.5x109 pfu/ml and low (ii) 0.5x109 pfu/ml doses. Mean arterial blood pressure (MBP), biochemical and histological assessments of maternal kidney, placenta and embryos were performed. RESULTS: Ad-sFlt-1 significantly increased MBP and induced severe glomerular endotheliosis in PlGF-/- mice at E10.5 gestation compared to wild-type animals. High sFlt-1 also significantly elevated albumincreatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury.At a high dose of sFlt-1, there was complete fetal resorption in the pregnant PlGF-/- mice, and even the lower dose of sFlt-1 induced severe fetal resorption and abnormal placental vascularization. Hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria in Ad-sFlt-1 treated pregnant PlGF-/- mice. To determine if placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/- placentas and embryos were generated in wild-time dams and exposed to high sFlt-1 environment. This resulted in reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF-/- mice. CONCLUSIONS: Placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 in preeclampsia and the hydrogen sulphide pathway may rescue preeclampsia phenotypes.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Carbon monoxide inhibits sprouting angiogenesis and vascular endothelial growth factor receptor-2 phosphorylation

Carbon monoxide (CO) is a gaseous autacoid known to positively regulate vascular tone; however, its role in angiogenesis is unknown. The aim of this study was to investigate the effect of CO on angiogenesis and vascular endothelial growth factor (VEGF) receptor-2 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor- reduced Matrigel and treated with a CO-releasing molecule (CORM-2) or exposed to CO gas (250 ppm). Here, we report the surprising finding that exposure to CO inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell actin reorganisation, cell proliferation, migration and capillary-like tube formation. Similarly, CO suppressed VEGF-mediated phosphorylation of VEGFR-2 at tyrosine residue 1175 and 1214 and basic fibroblast growth factor- (FGF-2) and VEGF-mediated Akt phosphorylation. Consistent with these data, mice exposed to 250 ppm CO (1h/day for 14 days) exhibited a marked decrease in FGF-2-induced Matrigel plug angiogenesis (p<0.05). These data establish a new biological function for CO in angiogenesis and point to a potential therapeutic use for CO as an anti-angiogenic agent in tumour suppression.