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• W2017709939 abstract "Skeletal muscle is one of the main physiological targets of insulin, a hormone that triggers a complex signaling cascade and that enhances the production of reactive oxygen species (ROS) in different cell types. ROS, currently considered second messengers, produce redox modifications in proteins such as ion channels that induce changes in their functional properties. In myotubes, insulin also enhances calcium release from intracellular stores. In this work, we studied in myotubes whether insulin stimulated ROS production and investigated the mechanisms underlying the insulin-dependent calcium increase: in particular, whether the late phase of the Ca2+ increase induced by insulin required ROS. We found that insulin stimulated ROS production, as detected with the probe 2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA). We used the translocation of p47phox from the cytoplasm to the plasma membrane as a marker of the activation of NADPH oxidase. Insulin-stimulated ROS generation was suppressed by the NADPH oxidase inhibitor apocynin and by small interfering RNA against p47phox, a regulatory NADPH oxidase subunit. Additionally, both protein kinase C and phosphatidylinositol 3-kinase are presumably involved in insulin-induced ROS generation because bisindolylmaleimide, a nonspecific protein kinase C inhibitor, and LY290042, an inhibitor of phosphatidylinositol 3-kinase, inhibited this increase. Bisindolylmaleimide, LY290042, apocynin, small interfering RNA against p47phox, and two drugs that interfere with inositol 1,4,5-trisphosphate-mediated Ca2+ release, xestospongin C and U73122, inhibited the intracellular Ca2+ increase produced by insulin. These combined results strongly suggest that insulin induces ROS generation trough NADPH activation and that this ROS increase is required for the intracellular Ca2+ rise mediated by inositol 1,4,5-trisphosphate receptors. Skeletal muscle is one of the main physiological targets of insulin, a hormone that triggers a complex signaling cascade and that enhances the production of reactive oxygen species (ROS) in different cell types. ROS, currently considered second messengers, produce redox modifications in proteins such as ion channels that induce changes in their functional properties. In myotubes, insulin also enhances calcium release from intracellular stores. In this work, we studied in myotubes whether insulin stimulated ROS production and investigated the mechanisms underlying the insulin-dependent calcium increase: in particular, whether the late phase of the Ca2+ increase induced by insulin required ROS. We found that insulin stimulated ROS production, as detected with the probe 2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA). We used the translocation of p47phox from the cytoplasm to the plasma membrane as a marker of the activation of NADPH oxidase. Insulin-stimulated ROS generation was suppressed by the NADPH oxidase inhibitor apocynin and by small interfering RNA against p47phox, a regulatory NADPH oxidase subunit. Additionally, both protein kinase C and phosphatidylinositol 3-kinase are presumably involved in insulin-induced ROS generation because bisindolylmaleimide, a nonspecific protein kinase C inhibitor, and LY290042, an inhibitor of phosphatidylinositol 3-kinase, inhibited this increase. Bisindolylmaleimide, LY290042, apocynin, small interfering RNA against p47phox, and two drugs that interfere with inositol 1,4,5-trisphosphate-mediated Ca2+ release, xestospongin C and U73122, inhibited the intracellular Ca2+ increase produced by insulin. These combined results strongly suggest that insulin induces ROS generation trough NADPH activation and that this ROS increase is required for the intracellular Ca2+ rise mediated by inositol 1,4,5-trisphosphate receptors. A transient intracellular Ca2+ increase is a key component of the excitation-coupling mechanism in skeletal muscle cells. Intracellular Ca2+ can also increase in response to stimuli other than membrane potential depolarization, including hormones such as insulin. We have previously reported that in myotubes, the addition of insulin produces a fast intracellular Ca2+ concentration transient, which requires external Ca2+ and is inhibited by the l-type Ca2+ channel blocker nifedipine and by ryanodine (1Espinosa A. Leiva A. Pena M. Muller M. Debandi A. Hidalgo C. Carrasco M.A. Jaimovich E. J. Cell. Physiol.. 2006; 209: 379-388Google Scholar). Other reports show that the Ca2+ increase evoked by insulin in skeletal muscle fibers depends on Ca2+ influx and is related to Glut-4 translocation (2Lanner J.T. Katz A. Tavi P. Sandstrom M.E. Zhang S.J. Wretman C. James S. Fauconnier J. Lannergren J. Bruton J.D. Westerblad H. Diabetes.. 2006; 55: 2077-2083Google Scholar). As steep changes in insulin concentration are unlikely to occur physiologically, we decided to explore changes in intracellular Ca2+ after longer exposure to insulin. We further investigated whether ROS 3The abbreviations used are: ROS, reactive oxygen species; PKC, protein kinase C; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; fluo-3AM, fluo-3 acetoxymethyl ester; CM-H2DCFDA, 7′-dichlorofluorescein diacetate; DsRed, red fluorescent protein; BTK, Bruton's tyrosine kinase; PH, pleckstrin homology; GFP, green fluorescent protein; ROI, region of interest; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA; BIM, bisindolylmaleimide; IRS-1, insulin receptor substrate-1. play a role in this process. It is known that ROS modulate the activity of Ca2+ release channels (3Hidalgo C. Donoso P. Antioxid. Redox Signal.. 2008; 10: 1275-1312Google Scholar), and in particular, the activity of skeletal muscle ryanodine receptors is regulated by NADPH oxidase-dependent redox modifications (4Hidalgo C. Sanchez G. Barrientos G. Aracena-Parks P. J. Biol. Chem.. 2006; 281: 26473-26482Google Scholar). On the other hand, in target tissues, insulin is known to generate ROS, which participate in signaling processes triggered by the hormone (5Goldstein B.J. Mahadev K. Wu X. Zhu L. Motoshima H. Antioxid. Redox Signal.. 2005; 7: 1021-1031Google Scholar). There are many intracellular sources of ROS in mammalian cells, such as mitochondria, xanthine oxidase, and NADPH oxidase (6Kamata H. Hirata H. Cell. Signal.. 1999; 11: 1-14Google Scholar, 7Clempus R.E. Griendling K.K. Cardiovasc. Res.. 2006; 71: 216-225Google Scholar). We have reported the presence of NADPH oxidase subunits in myotubes (1Espinosa A. Leiva A. Pena M. Muller M. Debandi A. Hidalgo C. Carrasco M.A. Jaimovich E. J. Cell. Physiol.. 2006; 209: 379-388Google Scholar) and in adult skeletal muscle transverse tubules (4Hidalgo C. Sanchez G. Barrientos G. Aracena-Parks P. J. Biol. Chem.. 2006; 281: 26473-26482Google Scholar). Recently, the NADPH oxidase has been involved in modifications of tyrosine phosphates that shut down signals evoked by insulin in adipocytes (8Mahadev K. Motoshima H. Wu X. Ruddy J.M. Arnold R.S. Cheng G. Lambeth J.D. Goldstein B.J. Mol. Cell. Biol.. 2004; 24: 1844-1854Google Scholar). The phagocytic NADPH oxidase is composed of five subunits: two catalytic subunits located in the plasma membrane (p22phox and gp91phox) and three cytoplasmic subunits (p40phox, p47phox, and p67phox), plus the small GTP-binding protein rac-1. Upon activation, these subunits translocate to the plasma membrane to form the active enzyme (9Bedard K. Krause K.H. Physiol. Rev.. 2007; 87: 245-313Google Scholar). The regulatory p47phox subunit has an autoinhibitory domain; phosphorylation of p47phox in serine residues causes its translocation to the plasma membrane to form the active enzymatic complex. Activation of p47phox depends on several serine/threonine and tyrosine kinases such as protein kinase C (PKC) (10Bey E.A. Xu B. Bhattacharjee A. Oldfield C.M. Zhao X. Li Q. Subbulakshmi V. Feldman G.M. Wientjes F.B. Cathcart M.K. J. Immunol.. 2004; 173: 5730-5738Google Scholar, 11Talior I. Tennenbaum T. Kuroki T. Eldar-Finkelman H. Am. J. Physiol.. 2005; 288: E405-E411Google Scholar), c-Src (12Chowdhury A.K. Watkins T. Parinandi N.L. Saatian B. Kleinberg M.E. Usatyuk P.V. Natarajan V. J. Biol. Chem.. 2005; 280: 20700-20711Google Scholar), and phosphatidylinositol 3-kinase (PI3K) (13Frey R.S. Gao X. Javaid K. Siddiqui S.S. Rahman A. Malik A.B. J. Biol. Chem.. 2006; 281: 16128-16138Google Scholar). By inducing phosphorylation of its receptor on tyrosine residues, insulin initiates a well described signal transduction cascade (14Saltiel A.R. Kahn C.R. Nature.. 2001; 414: 799-806Google Scholar), which involves PI3K activation. Insulin can also activate phospholipase C and generate IP3 and diacylglycerol (DAG). These second messengers can induce Ca2+ release and PKC activation, respectively. The main goal of this work was to investigate the possible correlation between the increase in Ca2+ and ROS induced by insulin. We found that both PI3K and PKC are needed to activate NADPH oxidase to produce ROS and that ROS are involved in the delayed Ca2+ increase induced by insulin, which involves IP3 receptor (IP3R) activation by ROS. Reagents—Insulin, fluo-3 acetoxymethyl ester (fluo-3AM), Alexa Fluor 488 and Alexa Fluor 633, and chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) were purchased from Invitrogen. Bordetella pertussis toxin was obtained from Calbiochem. Xestospongin C was a gift from Dr. Jordi Molgò (Centre National de la Recherche Scientifique, Gifsur-Yvette, France). Antibodies against insulin receptor, p47phox and gp91phox, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-linked anti-rabbit and anti-mouse IgG were purchased from Pierce. A plasmid encoding DsRed (Clontech) was used as a transfection marker. The plasmids used encode the fusion protein between the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (BTK) and the enhanced green fluorescent protein (GFP) ((PH) BTK-GFP) or the (PHmut) BTK-GFP (punctual mutation R28C of the PH domain). Both plasmids were kindly provided by Dr. Tamas Balla (National Institutes of Health, Bethesda, MD). A plasmid that encodes for HyPer protein was acquired from Evrogen Joint Stock Company, Moscow, Russia. Cell Culture—Primary cultured cells were obtained from rat neonatal hind limbs. The muscle tissue was mechanically dispersed and then incubated under mild agitation with 10% (w/v) collagenase for 15 min at 37 °C. The suspension was filtered through a Nytex (Sartorius, Göttingen, Germany) membrane and spun down at low speed, preplating was used to partially eliminate fibroblasts, and finally, cells were plated onto dishes (60 mm) with coverslips at a density of 0.6 × 106/dish. The culture medium used was Dulbecco's modified Eagle's medium/F-12, 10% bovine serum, 2.5% fetal calf serum, 100 mg/liter penicillin, 50 mg/liter streptomycin, and 2.5 mg/liter amphotericin B. To eliminate remaining fibroblasts, on the third day of culture, 10 μm cytosine arabinoside was added for 24 h. The medium was then replaced with serum-free medium. 6-8-day-old cultures were used. Ca2+ Measurement—Cytoplasmic Ca2+ images were obtained using inverted confocal microscopy (Carl Zeiss Pascal 5 LSM) from single, non-spontaneously contracting myotubes pre-loaded with fluo-3AM. Myotubes were washed with Krebs buffer (10 mm Hepes-Na, pH 7.4, 145 mm NaCl, 5 mm KCl, 2.6 mm CaCl2, 1 mm MgCl2, 5.6 mm glucose,) and loaded with 5.4 μm fluo-3AM (added from a stock solution in 20% pluronic acid-DMSO) for 30 min at room temperature. Cell-containing coverslips were mounted in a 1-ml capacity plastic chamber and placed in the microscope for fluorescence measurements (band pass 505-530 nm) after excitation with a 488 nm argon laser. Determination of ROS Production—ROS generation was determined in skeletal muscle cells using CM-H2DCFA. Myotubes were cultured on glass coverslips and incubated with 5 μm CM-H2DCFA for 15 min at 37 °C. Cells on coverslips were washed with PBS and placed at the bottom of an incubation chamber, which was transferred to the confocal microscope. CM-H2DCFA fluorescence was detected using excitation/emission wavelengths λexc/λem = 488/505-530 nm. In all measurements a control of the effect of laser excitation alone was performed. Noise in the images was removed by the Image J software (National Institutes of Health). Inmunofluorescence—Cells were placed on coverslips and fixed with methanol at -20 °C for 15 min. The blockade was performed in 1% bovine serum albumin for 60 min, and the primary antibodies were incubated overnight at 4 °C. Cells were washed and incubated with secondary antibody during 2 h at room temperature. Coverslips were mounted in VECTASHIELD (Vector Laboratories, Inc.) for confocal microscopy, and representative images were acquired. Negative controls used only secondary antibodies. Image Capture and Quantification of Fluorescence—Confocal image stacks were captured with a Zeiss LSM-5, Pascal 5 Axiovert 200 microscope, using LSM 5 3.2 image capture, analysis software, and a Plan-Apochromat ×63/1.4 oil differential interference contrast objective. Two-channel fluorescent image stacks (intensity I (x,y,z), voxel size Δx/Δy/Δz = 50/50/300 nm) were recorded in the multitrack mode. Channel-1 (Alexa Fluor 488) was used with excitation/emission wave-lengths λexc/λem = 488/505-530 nm, and channel-2 (Alexa Fluor 633) was used with λexc/λem = 635/>650 nm. We made sure that I (x,y,z) did not saturate and that the image background was slightly above zero by carefully adjusting the laser power, the detector gain, and the detector offset. Image stacks were deconvoluted with Huygens Scripting (Scientific Volume Imaging, Hilversum, Netherlands). All following image processing routines were developed in our laboratory on the basis of IDL (Interactive Data Language, ITT, Boulder, CO). To determine p47phox in the plasma membrane and the cytoplasm of myotubes, segmentations were performed to define different regions of interest (ROIs). First, the cross-section of myotubes was segmented by an intensity threshold in the green fluorescence channel (see Fig. 3D, center). Remaining holes inside the cells and artifacts outside the cells were filled or removed by morphological filters. Second, we used a border detection filter to identify the plasma membrane with a constant thickness of 400 nm (see Fig. 3D, right). The nuclear region was defined manually using the bright field image and excluded from the segmented cells. The fluorescence intensities of the cytoplasm and the plasma membrane were determined in the ROIs and divided by the respective surfaces (I/μm2). For all experiments, the protocols remained constant, and the quality of the segmentation was controlled interactively by overlaying the original fluorescent images with the segmented ROIs. Co-localization Analysis—A reliable segmentation of cellular structures containing p47phox or α-actinin could not be obtained by a simple threshold value in fluorescence intensity because background intensities vary within the cell thickness across the image. We solved this problem by applying gradient filters and selecting threshold values in the gradient histograms, which resulted in a homogeneous definition of p47phox or α-actinin signals (see Fig. 3A). For the quantification of co-localization between p47phox and α-actinin (Fig. 3A, left, ROI1/green regions and ROI2/red regions), we calculated the Manders co-localization coefficients M1 and M2 (15Manders E.M. Verbeek F.J. Aten J.A. J. Microsc. (Oxf.).. 1993; 1993: 375-382Google Scholar). M1 and M2 sum up the contribution of the respective fluorescence intensities in the co-localizing region ICh1/2(ROI1 ∩ ROI2), and we divided the number by the sum of the fluorescence intensities inside of the segmented regions ICh1(ROI1) or ICh2(ROI2). M1 and M2 can be interpreted directly as the amount of co-localizing p47phox/α-actinin labeled structures (Fig. 3A, lower, yellow regions) with respect to the total amount of segmented p47phox/α-actinin signals (Fig. 3A, lower left panel, yellow region plus green/red region). To reveal the significance of the co-localization coefficients, we implemented a defined displacement algorithm (16Sanchez G. Escobar M. Pedrozo Z. Macho P. Domenech R. Hartel S. Hidalgo C. Donoso P. Cardiovasc. Res.. 2008; 77: 380-386Google Scholar). Defined displacement algorithm shifts one fluorescent channel with respect to the second channel in a radial manner and calculates M1 and M2 successively for each displacement. When the radial displacement is larger than the size of the segmented ROIs, the calculated Manders coefficients quantify random scenarios of fluorescent structures inside the cellular borders, which were used to calculate the M1/M2 minima values in Fig. 3B. Data Analysis—All experiments were performed in at least three different cultures, and at least three cells per culture were measured in each case. Results are expressed as the mean ± S.E. Significance was evaluated using Student's t test for paired data and one-way analysis of variance followed by Dunnett's post hoc test for multiple column comparison to control column; p < 0.05 was considered significant. Insulin Induced ROS Generation through NADPH Oxidase Stimulation—Cultured myotubes preincubated with CM-H2DCFDA were stimulated with insulin to investigate insulin-induced ROS generation. Fig. 1A shows a representative experiment where images were acquired at different times after the addition of 50 nm insulin. This concentration was chosen because of its clear-cut effects on calcium transient generation (see below). The time dependence of the mean fluorescence increase mediated by the hormone is presented in Fig. 1B. A significant increase in intracellular ROS was evident 5 min after insulin stimulation. Cells incubated with apocynin, a specific inhibitor of p47phox-dependent NADPH oxidase (EC50 = 10 μm (7Clempus R.E. Griendling K.K. Cardiovasc. Res.. 2006; 71: 216-225Google Scholar)) displayed significantly reduced ROS production, and this effect was evident 5 min after insulin addition (Fig. 1B). To ascertain whether H2O2 is one of the ROS involved in this response, we used the HyPer protein, which is a genetically encoded fluorescent sensor for the specific detection of intracellular H2O2 (17Belousov V.V. Fradkov A.F. Lukyanov K.A. Staroverov D.B. Shakhbazov K.S. Terskikh A.V. Lukyanov S. Nat. Meth.. 2006; 3: 281-286Google Scholar). We measured the increase of HyPer fluorescence in myotubes after insulin stimulation. The increase in HyPer fluorescence was homogeneous in the cytoplasm of the myotube (Fig. 1C) and increased steadily in time, reaching a plateau of about 25-fold increase at 300 s; apocynin prevented this increase and even produced a small decrease of the initial endogenous levels of H2O2 (Fig. 1D). Role of the p47phox Subunit in ROS Generated by Insulin—The results obtained with apocynin strongly suggest that a p47phox-dependent NADPH oxidase is involved in ROS generation. To test this hypothesis, we measured insulin-dependent CM-H2DCFDA fluorescence in cells transfected with siRNA against p47phox, using siRNA with a random sequence as control (Fig. 2A). To identify the transfected cells, they were co-transfected with a plasmid encoding DsRed protein. Red-labeled cells, transfected with the random sequence, responded with a fluorescence increase 10 min after insulin stimulation (Fig. 2A). In contrast, red-labeled cells transfected with siRNA against p47phox did not respond to insulin, and the fluorescence signal was similar to that of cells expressing random siRNA in the basal state (Fig. 2, A and B). To evaluate whether p47phox expression was decreased in myotubes transfected with siRNA, we performed an immunofluorescence study against the p47phox subunit. The p47phox subunit was not detected in cells transfected with siRNA p47phox (Fig. 2C). To study the insulin-dependent activation of NADPH oxidase, we determined by immunofluorescence the translocation of p47phox. In basal conditions, p47phox showed a particular distribution in myotubes; the label was present in striations and displayed a significant co-localization with α-actinin, a Z line marker protein (Fig. 3, A and B). In the first minute after insulin addition, some p47phox label already appeared near the membrane (Fig. 3C, arrow). 15 min after stimulation, most of the label appeared in the periphery of myotubes, probably representing the location of the enzyme near the plasma membrane. At 60 min, the distribution of the fluorescence signal showed a pattern similar to the basal condition prior to insulin addition (Fig. 3C). We measured fluorescence intensity in regions of the cytoplasm and near the plasma membrane (Fig. 3D). We defined the region near the plasma membrane with a constant diameter of 400 nm and the region inside the cytoplasm excluding cell nuclei (Fig. 3D, right and center). In the region near the plasma membrane, a significant fluorescence increase was detected after insulin stimulation (Fig. 3E). This membrane location was seen only in stimulated sections, never in basal conditions (Fig. 3C shows different times). Cytoplasmic fluorescence decreases upon stimulation (Fig. 3E) concomitant to the increase in near membrane fluorescence; as both myotubes (basal and stimulated) were measured in identical conditions, these observed differences suggest p47phox translocation to the membrane region. Role of PI3K in ROS Generation—PI3K activation is a key event in insulin-dependent signal transduction, so we studied whether ROS generation depends on PI3K activity. Using a plasmid that encodes a fusion protein between the PH domain of BTK and the enhanced GFP ((PH)-BTK-GFP), we monitored the activation of PI3K as changes in probe fluorescence, considering that when PI3K is activated, it can recruit proteins with the PH domain to membranes. Cells transfected with (PH)-BTK-GFP showed basal fluorescence in nucleus and cytoplasm; 10 s after insulin stimulation, a distinct fluorescence increase appeared near the plasma membrane and in regions that may correspond to transverse tubules. This particular fluorescence increase disappeared after 90 s (Fig. 4A). These results indicate that activation of PI3K is an early event of the signal transduction cascade triggered by insulin that occurs prior to ROS generation. We next measured ROS generation induced by 50 nm insulin 15 min after stimulation in controls or in cells preincubated with different drugs. ROS production was completely prevented by genistein, an inhibitor of insulin receptor (Fig. 4B). LY290042, an inhibitor of the PI3K pathway, also produced a complete inhibition of fluorescence increase with respect to the control stimulated condition (Fig. 4B), suggesting that PI3K is a key player in ROS generation triggered by insulin. Previous reports suggest that PKB can phosphorylate p47phox. To probe the role of PKB in ROS generation, we used a PKB inhibitor, PKBI (Fig. 4C, PKBI). We measured ROS generation 30 min after insulin stimulation and found similar values for ROS production both in the presence of inhibitor and in control conditions (Fig. 4C). Accordingly, we can conclude that insulin-stimulated ROS generation is dependent on PI3K but not on PKB. To evaluate the participation of PI3K in the activation of NADPH oxidase, we studied the insulin-dependent translocation of p47phox to the plasma membrane in the presence of LY290042. As shown above, p47phox is present in the cytoplasm in basal conditions; after 10 min of insulin stimulation in the presence of LY290042, most of the label still remained in striations (Fig. 4C). There was some translocation in the presence of the inhibitor but clearly much less than in the control conditions illustrated in Fig. 3C. Role of PKC in ROS Generation—To study the possible role of PKC in insulin-dependent ROS generation, we preincubated myotubes with BIM, a general inhibitor of PKC. Incubation with BIM inhibited insulin-induced ROS generation (Fig. 5A). In addition, 0.5 μm BIM inhibited the increase in HyPer fluorescence induced by insulin (Fig. 5C) and also prevented the translocation of p47phox to the plasma membrane induced by insulin (Fig. 5B). Insulin Increased the Intracellular Ca2+ Concentration in Cultured Myotubes—We had some evidence for delayed Ca2+ oscillations occurring after the fast Ca2+ transient induced by insulin in myotubes (1Espinosa A. Leiva A. Pena M. Muller M. Debandi A. Hidalgo C. Carrasco M.A. Jaimovich E. J. Cell. Physiol.. 2006; 209: 379-388Google Scholar); these oscillations were seen at insulin concentrations as low as 1-10 nm but were consistently larger (80-fold increase in fluo-3 fluorescence) at 50 nm insulin. Accordingly, in this report, we performed a detailed study of these delayed Ca2+ signals in cultured myotubes using the fluorescence probe fluo-3AM. The insulin-dependent Ca2+ signals studied in this work are independent of the fast Ca2+ transients that require extracellular Ca2+ and functional ryanodine receptors (1Espinosa A. Leiva A. Pena M. Muller M. Debandi A. Hidalgo C. Carrasco M.A. Jaimovich E. J. Cell. Physiol.. 2006; 209: 379-388Google Scholar). Thus, in conditions in which the fast transient is absent (i.e. in the absence of external Ca2+), the delayed signal remained unchanged, whereas inhibitors of the slow Ca2+ signal (see below) did not affect the fast Ca2+ signal (for example, Fig. 6C). We investigated the possible role of NADPH oxidase-generated ROS on the slower Ca2+ signals produced by insulin. To that aim, we measured the effect of both diphenyliodonium (Fig. 6A, DPI) and apocynin on insulin-evoked signals. Preincubation of myotubes with both inhibitors abolished the insulin-dependent intracellular Ca2+ increases (Fig. 6A). On the same line, we studied the role of NADPH oxidase using siRNA against p47phox. Fig. 6B shows a representative response of transfected myotubes in which the Ca2+ increase produced by insulin was reduced significantly in myotubes transfected with siRNA against p47phox. In addition, the PKC inhibitor BIM decreased significantly the insulin-dependent Ca2+ increase, whereas the PI3K inhibitors wortmannin and LY290042 completely prevented this response (Fig. 6C). To test the involvement of IP3 receptors on Ca2+ signals, we preincubated the myotubes with two inhibitors of the IP3 pathway, U73122 and xestospongin C; the insulin-stimulated Ca2+ increase was abolished in both conditions (Fig. 6D). In this work, we provide evidence favoring a role for insulin in ROS generation due to NADPH oxidase activation in skeletal muscle cells. We show, in addition, that enhanced ROS generation forms part of the signaling mechanisms whereby insulin increases intracellular Ca2+ concentration. The fast increase of intracellular ROS produced by insulin was detected using both CM-H2DCFDA as fluorescence probe and a molecular recombinant protein that in vitro displays half-maximal increase in fluorescence at 10 μm H2O2. CM-H2DCFDA is a probe susceptible to oxidation by H2O2 and other ROS and nitric oxide-derived species, but transfection of myotubes with HyPer plasmid provided us with a specific tool to ascertain that 50 nm insulin produced H2O2. An increase in intracellular peroxide concentration was detected with both probes as early as 1 min after insulin addition. In various cell types, the NADPH oxidase has been described as the intracellular source of H2O2 (9Bedard K. Krause K.H. Physiol. Rev.. 2007; 87: 245-313Google Scholar). This enzyme has defensive functions in phagocytic cells, where it catalyzes the one-electron reduction of oxygen to release superoxide anion that rapidly dismutates to H2O2, a microbicide agent. However, in non-phagocytic cells, H2O2 is considered a second messenger due to its capacity to produce reversible post-translational oxidative modification of proteins (18Rhee S.G. Science.. 2006; 312: 1882-1883Google Scholar). We have reported previously that myotubes possess NADPH oxidase activity that is stimulated by membrane depolarization (1Espinosa A. Leiva A. Pena M. Muller M. Debandi A. Hidalgo C. Carrasco M.A. Jaimovich E. J. Cell. Physiol.. 2006; 209: 379-388Google Scholar). Here, we provide evidence indicating that insulin also stimulates a p47phox-dependent NADPH oxidase in skeletal muscle cells, as indicated by our findings that insulin-stimulated ROS generation was inhibited in myotubes preincubated with apocynin or transfected with an siRNA against p47phox. In addition, our current results, showing p47phox translocation from cytoplasmic striations to the plasma membrane from the first minute after stimulation with insulin (Fig. 3C), provide further support for NADPH oxidase activation by insulin. Ca2+ signals mediate a variety of physiological processes in skeletal muscle cells; in particular, muscle depolarization elicits fast Ca2+ signals that initiate muscle contraction and slower signals that regulate gene expression (19Jaimovich E. Espinosa A. Biol. Res.. 2004; 37: 625-633Google Scholar). Insulin-dependent Ca2+ influx has been implicated in glucose uptake in adult muscle fibers (2Lanner J.T. Katz A. Tavi P. Sandstrom M.E. Zhang S.J. Wretman C. James S. Fauconnier J. Lannergren J. Bruton J.D. Westerblad H. Diabetes.. 2006; 55: 2077-2083Google Scholar); in that case, a delayed effect (after 20 min) of insulin that required external Ca2+ was reported. A direct link between NADPH-mediated ROS production and glucose uptake seems unlikely because a recent report showed no inhibition of glucose uptake by apocynin in insulin-stimulated skeletal muscle cell lines and primary cultures (20Hutchinson D.S. Csikasz R.I. Yamamoto D.L. Shabalina I.G. Wikstrom P. Wilcke M. Bengtsson T. Cell. Signal.. 2007; 19: 1610-1620Google Scholar). Insulin has been involved in the regulation of the expression of a number of skeletal muscle genes related to carbohydrate metabolism and protein synthesis (21Rome S. Clement K. Rabasa-Lhoret R. Loizon E. Poitou C. Barsh G.S. Riou J.P. Laville M. Vidal H. J. Biol. Chem.. 2003; 278: 18063-18068Google Scholar). In skeletal muscle cells, Ca2+ release from IP3-dependent intracellular stores mediates the regulation of signaling cascades, leading to expression or repression of both early and late genes (22Powell J.A. Carrasco M.A. Adams D.S. Drouet B. Rios J. Muller M. Estrada M. Jaimovich E. J. Cell Sci.. 2001; 114: 3673-3683Google Scholar, 23Carrasco M.A. Riveros N. Rios J. Muller M. Torres F. Pineda J. Lantadilla S. Jaimovich E. Am. J. Physiol.. 2003; 284: C1438-C1447Google Scholar, 24Juretic N. Urzua U. Munroe D.J. Jaimovich E. Riveros N. J. Cell. Physiol.. 2007; 210: 819-830Google Scholar). IP3 receptors are highly dependent on its regulators and modulators (25Choe C.U. Ehrlich B.E. Science's STKE 2006.2006: re15Google Scholar). A redox sensor protein has been shown to regulate type 1 IP3R (26Mikoshiba K. J. Neurochem.. 2007; 102: 1426-1446Google Scholar), redox-sensitive free thiol groups have been identified in the IP3R molecule (27Joseph S.K. Nakao S.K. Sukumvanich S. Biochem. J.. 2006; 393: 575-582Google Scholar), and direct redox modifications of isolated IP3R channels have been described (28Kaplin A.I. Ferris C.D. Voglmaier S.M. Snyder S.H. J. Biol. Chem.. 1994; 269: 28972-28978Google Scholar). Accordingly, the Ca2+ signals elicited by insulin, which require NADPH oxidase and IP3 receptor activation, may also be involved in regulation of gene expression. Further studies are needed to elucidate this point. We have previously shown that electrical activity stimulates ROS production by NADPH oxidase in skeletal muscle cells and prompts the emergence of Ca2+ signals due to ROS-stimulated ryanodine receptors (1Espinosa A. Leiva A. Pena M. Muller M. Debandi A. Hidalgo C. Carrasco M.A. Jaimovich E. J. Cell. Physiol.. 2006; 209: 379-388Google Scholar). Additionally, NADPH oxidase-generated ROS stimulate ryanodine receptor-mediated Ca2+ release from triad-enriched vesicles isolated from adult skeletal muscle cells (4Hidalgo C. Sanchez G. Barrientos G. Aracena-Parks P. J. Biol. Chem.. 2006; 281: 26473-26482Google Scholar). As electrical activity is a constant feature of functional muscle, insulin and electrical activity may jointly stimulate ROS generation, which by enhancing Ca2+ release mediated by ryanodine or IP3 receptors, as shown in this work, generates Ca2+ signals that regulate muscle contraction or gene expression. In summary, we propose the following mechanism for insulin-dependent ROS production and delayed Ca2+signal generation (Fig. 7). Insulin binding to its receptor promotes phosphorylation of its β subunit in tyrosine residues and recruits IRS-1; PI3K activation resulting from its binding to IRS-1 produces inositol 1,4,5-trisphosphate, which stimulates phospholipase C γ to produce IP3 and DAG. The increased DAG production at the plasma membrane activates PKCδ, and this enzyme phosphorylates p47phox, allowing this subunit to migrate to the membrane to form the activated NADPH oxidase complex; the resulting ROS modify IP3 receptors and facilitate IP3-induced Ca2+ release, producing the delayed Ca2+ signals induced by insulin (Fig. 7). Although insulin-enhanced Ca2+ release can certainly generate Ca2+ signals of physiological relevance on their own, insulin-derived H2O2 generation can also play an important role in muscle function. In fact, H2O2 as a messenger has been linked to the regulation of various redox-sensitive proteins, among them the ryanodine receptors (3Hidalgo C. Donoso P. Antioxid. Redox Signal.. 2008; 10: 1275-1312Google Scholar, 29Feng W. Liu G. Allen P.D. Pessah I.N. J. Biol. Chem.. 2000; 275: 35902-35907Google Scholar). Our results propose a new aspect of insulin-initiated signal transduction in skeletal muscle cells. We postulate an unexplored relationship between H2O2 production by insulin-stimulated NADPH oxidase and Ca2+ release mediated by IP3 receptors, which may underlie insulin-dependent regulation of gene expression in muscle cells." @default.
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• W2017709939 title "NADPH Oxidase and Hydrogen Peroxide Mediate Insulin-induced Calcium Increase in Skeletal Muscle Cells" @default.
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