Arginine vasopressin increases iNOS-NO system activity in cardiac fibroblasts through NF-kappaB activation and its relation with myocardial fibrosis

Previous studies have shown that arginine vasopressin (AVP) promotes myocardial fibrosis (MF), whereas nitric oxide (NO) inhibits MF. Cardiac fibroblasts (CFs) are the main target cells of MF. However, the modulatory effect of AVP on NO production in CFs and the role of this effect in MF are still unknown. In the present study, CFs obtained from Sprague-Dawley rats were stimulated with or without AVP and pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor kappa-B (NF-kappaB). NO production and NOS activity were detected with absorption spectrometry, inducible nitric oxide synthase (iNOS) protein with Western blot analysis, iNOS mRNA with real-time PCR, CF collagen synthesis with [(3)H]proline incorporation, and NF-kappaB activation with immunofluorescence staining and Western blot analysis. The results showed that AVP increased NO production in a dose- and time-dependent manner, with maximal effects at 10(-7) mol/l after 24-h stimulation. AVP also increased NOS activity, protein and mRNA levels of iNOS in a coincident manner. Furthermore, AVP also increased CF collagen synthesis in a dose- and time-dependent manner. In addition, it was found that NF-kappaB was activated by AVP, and that PDTC could inhibit NO production, NOS activity, protein and mRNA levels of iNOS stimulated by AVP in a dose-dependent manner. The inhibitory effects of PDTC on NF-kappaB translocation were coincident with the effects of PDTC on iNOS-NO system activity. It is suggested that AVP increases NO production via the regulation of iNOS gene expression, and the upregulation of iNOS gene expression stimulated by AVP is mediated through NF-kappaB activation. NO production induced by AVP may counteract the profibrotic effects of AVP, thus the development of MF perhaps depends on the balance between profibrotic AVP and antifibrotic NO effects on MF.     

Chronic stimulation of glucose transporter gene expression in L6 myocytes mediated via the insulin-like growth factor-1 receptor

We have used differentiated L6 myocytes to investigate the regulation of glucose transporter gene expression by insulin and insulin-like growth factor-1 (IGF-1). Chronic exposure to insulin (1 microM) or IGF-1 (10 nm) resulted in a 2- to 5-fold stimulation of 3H-2-deoxy-D-glucose uptake and a corresponding increase in the expression of rat brain/HepG2-type glucose transporter mRNA (GTmRNA) and immunoreactive transporter protein. The dose responses to both insulin and IGF-1 for stimulation of glucose uptake were paralleled by the expression of GTmRNA. Glucose uptake and GTmRNA levels were half maximally stimulated by 350 and 100 nM insulin, respectively, or by 2 nM IGF-1. Comparison of receptor occupancy with stimulation of glucose uptake and GTmRNA expression suggests that insulin exerts its effects through the IGF-1 receptor. Fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and phorbol ester had little or no effect on GTmRNA expression. These results demonstrate that the IGF-1 receptor mediates chronic regulation of transporter mRNA expression and protein synthesis and activity in cultured rat muscle cells.     

Effects of insulin and insulin-like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin-like growth factor 1 (IGF-1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF-1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF-1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF-1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF-1 promote osteoblast differentiation and mineralization in vitro, IGF-1, but not insulin, can induce osteoblast proliferation. The IGF-1-induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF-1-mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3-kinase inhibitor. Osteocalcin, an osteoblast-specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose- and time-dependent manner after insulin treatment, whereas it was decreased with IGF-1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF-1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis.     

Angiotensin II elevates nitric oxide synthase 3 expression and nitric oxide production via a mitogen-activated protein kinase cascade in ovine fetoplacental artery endothelial cells

Normal pregnancy is associated with high angiotensin II (ANG II) concentrations in the maternal and fetal circulation. These high levels of ANG II may promote production vasodilators such as nitric oxide (NO). ANG II receptors are expressed in ovine fetoplacental artery endothelial (OFPAE) cells and mediate ANG II-stimulated OFPAE cell proliferation. Herein, we tested whether ANG II stimulated NO synthase 3 (NOS3, also known as eNOS) expression and total NO (NO(x)) production via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also known as ERK1/2) in OFPAE cells. ANG II elevated (P < 0.05) eNOS protein, but not mRNA levels with a maximum effect at 10 nM. ANG II also dose dependently increased (P < 0.05) NO(x) production with a maximal effect at doses of 1-100 nM. Activation of ERK1/2 by ANG II was determined by immunocytochemistry and Western blot analysis. ANG II rapidly induced positive staining for phosphorylated ERK1/2, appearing in cytosol after 1-5 min of ANG II treatment, accumulating in nuclei after 10 min, and disappearing at 15 min. ANG II increased (P < 0.05) phosphorylated ERK1/2 protein levels. Activation of ERK1/2 was confirmed by an immunocomplex kinase assay using ELK1 as a substrate. PD98059 significantly inhibited ANG II-induced ERK1/2 activation, and the ANG II-elevated eNOS protein levels but only partially reduced ANG II-increased NO(x) production. Thus, in OFPAE cells, the ANG II increased NO(x) production is associated with elevated eNOS protein expression, which is mediated at least in part via activation of the mitogen-activated protein kinase kinase1 and kinase2 (MAP2K1 and MAP2K2, known also as MEK1/2)/ERK1/2 cascade. Together with our previous observation that ANG II stimulates OFPAE cell proliferation, these data suggest that ANG II is a key regulator for both vasodilation and angiogenesis in the ovine fetoplacenta.     

Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia.     

Insulin restores glucose inhibition of adenosine transport by increasing the expression and activity of the equilibrative nucleoside transporter 2 in human umbilical vein endothelium

L-Arginine transport and nitric oxide (NO) synthesis (L-arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D-glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D-glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2-, but lower hENT1-mediated transport in normal D-glucose. Insulin increased hENT2 protein abundance in normal or high D-glucose, but reduced hENT1 protein abundance in normal D-glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D-glucose. Insulin effect on hENT1 mRNA expression in normal D-glucose was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and mimicked by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). L-NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO-independent mechanism. These findings could be important in hyperglycemia-associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development.     

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.     

AMPK activation inhibits the expression of HIF-1alpha induced by insulin and IGF-1

Insulin, insulin like growth factor (IGF)-1, and AMP-activated protein kinase (AMPK) signaling regulate independently angiogenesis through vascular endothelial growth factor (VEGF) expression. In the present study, we investigated a potential cross-talk between these signaling pathways on hypoxia-inducible factor (HIF)-1alpha and VEGF expression. Retinal epithelial ARPE-19 cells were treated with AICAR, an AMPK activator, alone or in combination with insulin and IGF-1. AICAR stimulated VEGF mRNA expression, but did not modify the insulin- and IGF-1-induced VEGF expression. We have investigated the effect of AICAR on insulin and IGF-1 signaling pathways. We observed that AICAR increased insulin- and IGF-1-induced phosphorylation of PKB, whereas phosphorylation of S6K-1 was decreased. Moreover, AICAR and metformin inhibited the ability of insulin and IGF-1 to induce HIF-1alpha expression. These results show that AICAR and insulin/IGF-1 regulate VEGF expression through different mechanisms.     

Endogenous nitric oxide proves not to be involved in the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells.

Combined treatment of isolated rat gastric mucosal (RGM1) cells with interleukin (IL)-1beta and transforming growth factor (TGF)-alpha resulted in expression of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein 24 hr after the treatment. Constitutive NOS (nNOS) protein was not proved in the cells and not activated by IL-1beta+TGFalpha. Although IL-1beta and TGF-alpha alone exerted little or no effect on NO2 production, their combination gradually increased NO2- production from 12 to 24 hr following treatment. NO2- production stimulated by IL-1beta + TGFalpha was significantly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, yet not by D-NAME. S-nitroso-N-acetyl-D,L-penicillamine and sodium nitropruside significantly inhibited both spontaneous and TGF-alpha stimulated DNA synthesis. Nonetheless, L-NAME did not affect the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells, eliminating the possibility of involvement of NO in the underlying mechanisms.     

Carbon monoxide decreases the level of iNOS protein and active dimer in IL-1β-stimulated hepatocytes

There is evidence that NO can regulate CO production, however less is known about CO regulation of NO synthesis. Our studies were undertaken to define how CO regulates iNOS in cultured hepatocytes. CO (250 ppm) exposure resulted in a significant decrease in iNOS protein, nitrite production, level of active iNOS dimer and cytosolic iNOS activity in cells stimulated with cytokines (IL-1β) or transfected with the human iNOS gene. However, IL-1β-stimulated iNOS mRNA expression was unaffected by CO. These effects of CO on iNOS protein levels were inhibited when CO was scavenged using hemoglobin. HO-1 induction with an adenoviral vector carrying HO-1 showed a decrease in total iNOS protein, nitrite production, and iNOS dimer level from cells stimulated by IL-1β. iNOS protein level was significantly higher in lung endothelial cells isolated from HO-1 knockout mice compared to wild type cultures stimulated with cytokines mixture. CO was found to increase p38 phosphorylation and p38 inhibition using SB203580 increased iNOS protein levels in response to IL-1β. Interestingly, proteasome inhibitors (MG132 and Lactacystin) and an autophagy inhibitor (3-methyladenine) reversed CO influence iNOS levels. Our results imply that CO exposure decreases NO production by suppressing dimer formation and increasing iNOS degradation through a process involving p38 activation.     

Antagonic effects of oestradiol in interaction with IGF-1 on proliferation of lactotroph cells in vitro

The effects of IGF-1, 17 β oestradiol and its functional interaction on lactotrophs cell proliferation were evaluated. In addition we investigated the involvement of PKC α, ɛ and phosphorilated ERK, in the mitogenic process. Primary cell cultures of adenohypophysis from female Wistar rats were studied in serum free conditions. The proliferation of lactotrophs was determined by double immunostaining for BrdU and PRL. The incubation with IGF-1 5, 30 or 100 ng/ml during 48 or 72 h increased lactotrophs proliferation two–threefold depending on IGF-1 concentration. Co-incubation of IGF-1 (30 ng/ml) with genistein (25 μM) or BIM (0.5 or 2 μM), lowered of tyrosine kinase receptor or of PKC respectively, inhibited the induced IGF-1 lactotrophs proliferation. 17 β oestradiol (1, 10 or 100 nM) had not mitogenic effect, whereas in the presence of serum PRL cells proliferation was stimulated. Co-incubation with 1 nM oestradiol and IGF-1 significantly decreased the lactotroph BrdU-labelling achieved with IGF-1. PKC α, ɛ and ERK1/2 levels measured by western blot augmented in the presence of IGF-1 and were inhibited with the addition of genistein, supporting a participation of these enzymes in the proliferate process. Co-incubation of IGF-1 with 1 nM oestradiol decreased both PKC isoforms and activated ERK1/2 levels, suggesting that oestradiol would exert its antiproliferative effect by acting on the signalling pathway of IGF-1. The results revealed antagonic effects of oestradiol on lactotroph proliferation depending on its concentration and the presence of IGF-1.     

Inability of insulin and insulinlike growth factor-1 to stimulate sugar or amino acid transport and thymidine incorporation in cultured myeloma cells.

NS-1 mouse plasmacytoma cells were examined for their insulin and insulinlike growth factor-1 (IGF-1) binding characteristics and ability to produce peptide-dependent cellular effects. At concentrations of labelled insulin (i.e., 1.7 x 10(-10) M) or IGF-1 (i.e., 1.5 x 10(-10) M), NS-1 cells specifically bind 0.2 +/- 0.06 fmol insulin per 10(6) cells (n = 7), where little, if any, IGF-1 specific binding was observed (0.02 +/- 0.01 fmol/10(6) cells) (n = 3). Additionally, the data indicate that the total number of insulin binding sites per cell was 3200 +/- 390 (n = 3). Insulin was employed at various concentrations (6.7-667 nM) and failed to stimulate either sugar or amino acid transport. Insulin at low concentrations (i.e., 6.7 or 67 nM) did not stimulate DNA synthesis, yet a small but significant increase was observed at a concentration of 667 nM insulin. IGF-1 did not stimulate DNA synthesis at all concentrations employed (1.4-143 nM). In summary, there exists a small but significant number of insulin receptors, little insulin-stimulated DNA synthesis, and no apparent insulin stimulation of sugar or amino acid transport. Also, since there is no significant IGF-1 binding and no IGF-1 stimulation of DNA synthesis, these findings indicate that this cell line might be a good candidate for the study of insulin receptor function as a transfection recipient of insulin receptor genes.     

Regulation of the nitric oxide system in human adipose tissue

Nitric oxide (NO) is involved in adipose tissue biology by influencing adipogenesis, insulin-stimulated glucose uptake, and lipolysis. The enzymes responsible for NO formation in adipose cells are endothelial NO synthase (eNOS) and inducible NO synthase (iNOS), whereas neuronal NO synthase (bNOS) is not expressed in adipocytes. We characterized the expression pattern and the influence of adipogenesis, obesity, and weight loss on genes belonging to the NO system in human subcutaneous adipose cells by combining in vivo and in vitro studies. Expression of most of the genes known to belong to the NO system (eNOS, iNOS, subunits of the soluble guanylate cyclase, and both genes encoding cGMP-dependent protein kinases) in human adipose tissue and isolated human adipocytes was detected. In vitro adipogenic differentiation increased the expression level of iNOS significantly, whereas eNOS expression levels were not influenced. The genes encoding eNOS, iNOS, and cGMP-dependent protein kinase 1 were expressed at higher levels in obese women. Expression of these genes, however, was not influenced by 5% weight loss. Insulin and angiotensin II (Ang II) increased NO production by human preadipocytes in vitro. Increased eNOS and iNOS expression in adipocytes and local effects of insulin and Ang II may increase adipose tissue production of NO in obesity.     

Coupling of insulin-responsive glucose transport to receptors for insulin-like growth factor 1 in primary human fibroblasts

We have recently described an insulin-resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine----proline mutation at amino acid position 233 in the alpha-chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2-deoxyglucose uptake by insulin and insulin-like growth factor 1 (IGF-1). Insulin hardly stimulates basal 2-deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2-deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF-1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF-1. With both types of fibroblasts, maximal IGF-1 response is reached at about 10 nM IGF-1, the ED50 being approximately 4 nM. The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF-1. Insulin binds with an approximately 200-fold lower affinity to IGF-1 receptors, compared to homologous IGF-1 binding. As an insulin concentration of 10 microM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF-1, it seems that occupation of IGF-1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.     

Nitric oxide regulates prolidase activity by serine/threonine phosphorylation

Prolidase [E.C. 3.4.13.9], a member of the matrix metalloproteinase (MMP) family, is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. It plays an important role in collagen metabolism, matrix remodeling and cell growth. Nitric oxide (NO), a versatile signaling molecule, regulates many processes including collagen synthesis and matrix remodeling and, thereby, may modulate angiogenesis, tumor invasiveness, and metastasis. Thus, we considered that prolidase may be an important target of NO regulation. In our study, SIN I and DETA/NO were used as NO donors. Both donors increased prolidase activity in a time-dependent and dose-dependent manner. Prolidase activity increased not only with NO donors but also with endogenous NO in cells transfected with iNOS. The effect of iNOS was abolished by treatment with S-methylisothiourea (SMT), a selective inhibitor of iNOS. However, with either exogenous or endogenous sources of NO, the increase in prolidase activity was not accompanied by increased prolidase expression. Therefore, we suspected phosphorylation of prolidase as a potential mechanism regulating enzyme activation. We observed increased serine/threonine phosphorylation on prolidase protein in cells treated with NO donors and in cells transfected with iNOS. To determinate the pathways that may mediate prolidase induction by NO, we first used 8-Br-cGMP, a cGMP agonist, and found that 8-Br-cGMP strongly and rapidly stimulated prolidase activity accompanied by increased phosphorylation. Rp-8-Br-pCPT-cGMP, an inhibitor of cGMP, reduced NO donor-stimulated prolidase activity to control levels. To test whether the MAPK pathway is involved in this NO-dependent activation, we used an ERK1/2 inhibitor and found that it had no effect on prolidase activity increased by NO donors. These results demonstrate that NO stimulates prolidase activity by increasing serine/threonine phosphorylation through PKG-cGMP pathway, but independent of MAPK and suggest an interaction between inflammatory signaling pathways and regulation of the terminal step of matrix degradation.