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Potassium channel openers prevent palmitate-induced insulin resistance in C2C12 myotubes
Archives of Biochemistry and Biophysics 541 (2014) 47–52
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Potassium channel openers prevent palmitate-induced insulin resistance in C2C12 myotubes Dorota Dymkowska, Beata Drabarek, Justyna Jakubczyk, Sylwia Wojciechowska, Krzysztof Zabłocki ⇑ Nencki Institute of Experimental Biology, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 9 July 2013 and in revised form 28 October 2013 Available online 19 November 2013 Keywords: C2C12 myotubes Insulin resistance Nicorandil NS1619 Reactive oxygen species
a b s t r a c t Insulin resistance (IR) of muscle cells is an early symptom of type 2 diabetes. It often results from excessive lipid accumulation in muscle fibers which under in vitro experimental conditions may be induced by incubation of muscle cells with palmitate. IR is manifested as a reduced response of cells to insulin expressed by lowered Akt kinase phosphorylation and decreased insulin-dependent glucose uptake. Stimulation of mitochondrial oxidative metabolism by mild dissipation of the mitochondrial potential is thought to increase fatty acid utilization and thereby prevent insulin resistance. Here it is shown that nicorandil and NS1619, which are openers of two different mitochondrial potassium channels, protect C2C12 myotubes from palmitate-induced insulin resistance. Preincubation of myotubes with 5-hydroxydecanoate abolishes the protective effect of nicorandil. The efficient concentrations of both openers are far below those commonly applied for cytoprotection. This is probably why their effects on the mitochondrial energy metabolism are small. These data suggest that opening of mitochondrial potassium channels could be a promising approach in prevention and therapy of insulin resistance related to dyslipidemia and obesity. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Insulin resistance is an early symptom of type 2 diabetes, which may appear in apparently healthy young people. It gradually leads to increased blood glucose concentration, glucose intolerance, hyperinsulinemia and finally an impairment of the b-cell function. In many cases IR1 develops in obese individuals, and results from insufficient oxidation of fatty acids in muscle and liver cells. Accumulation of acylglycerol deposits in cells causes an excessive activation of protein kinase C (PKC) and multiple phosphorylation of insulin receptor substrate-1 (IRS-1) at serine residues that prevents its proper phosphorylation catalyzed by the intrinsic insulin receptor tyrosine kinase. In addition, the excessive fatty acid accumulation enhances insulin resistance via several intermediates such as acylCoA, diacylglycerol and ceramides affecting the insulin signaling pathway and glucose metabolism. Moreover, fatty-acid induced oxidative stress is suggested to be an important factor de-sensitizing cells to insulin [1]. The excessive generation of reactive oxygen spe⇑ Corresponding author. Address: Nencki Institute of Experimental Biology, Pasteura str, 02-093 Warsaw, Poland. E-mail address: [email protected] (K. Zabłocki). 1 Abbreviations used: IR, Insulin resistance; PKC, protein kinase C; IRS-1, insulin receptor substrate-1; ROS, reactive oxygen species; NEFA, non-esterified fatty acids; FBS, fetal bovine serum; PAGE, Polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; DFFH 2 -DA, difluorodihydrofluorescein diacetate; 5-HD, 5hydroxydecanoate. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.11.003
cies (ROS) is mostly attributed to the NADPH oxidase activity and the mitochondrial electron transport chain, but their relative contribution to the cellular oxidative stress is still under debate [2,3]. Nevertheless, the link between IR and mitochondrial dysfunction is widely accepted. It has been well documented that stimulation of fatty acid oxidation in the liver and muscle substantially improves the insulin sensitivity of these tissues [4,5]. In line with that observation, it has also been shown that dissipation of mitochondrial membrane potential (DW with mitochondrial uncouplers stimulates fatty acid oxidation and potentiates insulin-stimulated glucose uptake by human cultured myotubes, indicating enhanced insulin sensitivity of these cells [6]. Conversely, inhibition of the respiratory chain with oligomycin and/or antimycin reduces fatty acid oxidation and decreases the insulin sensitivity of myotubes. Mitochondrial uncoupling not only stimulates fatty acid oxidation (restoring insulin sensitivity) but also reduces the cellular energy charge, activating AMPK. Eventually it enhances muscle glucose uptake and limits hepatic gluconeogenesis [7]. Nicorandil is a nitrate derivative of nicotinamide, thus it exerts vasodilatory effects due to vascular smooth muscle relaxation. In addition, it was found to activate the ATP-sensitive K+ channel in cardiac muscle mitochondria. The latter effect is interpreted as a mechanism of pharmacologically induced heart preconditioning based on K+ cycling across the inner mitochondrial membrane and mild mitochondrial membrane depolarization. It prevents Ca2+ overloading of mitochondria and protects cells against
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ischemia-induced injury [8]. The decrease in the mitochondrial membrane potential resulting from the enhanced K+ permeability of the inner mitochondrial membrane correlates with an accelerated oxygen consumption by muscle cells or isolated mitochondria treated with nicorandil [9]. Therefore, the stimulation of respiration following pharmacological opening of mitochondrial potassium channels could be a remedy for insulin resistance, restoring the proper cellular response to this hormone. Data shown here indicate that two potassium channel openers of different specificity (nicorandil and NS1619 activating an ATPsensitive channel, KATP, and the calcium-dependent large-conductance BK channel, respectively) prevent palmitate-induced insulin resistance in C2C12 myotubes. This finding confirms the assumption presented above and indicates that potassium channel openers may be useful in antidiabetic therapy. Furthermore, only mild changes in the mitochondrial metabolism induced by these compounds suggest that even a marginal increase of the mitochondrial inner membrane permeability for potassium is sufficient to prevent insulin resistance. This latter finding together with only slightly increased ROS generation indicate that the mechanism involving potassium channel openers in the prevention of palmitate-induced IR of myotubes is not straightforward. Probably it is not a simple consequence of mitochondrial malfunctioning, but relies on a complex, as yet unidentified regulatory processes. Material and methods Preparation of palmitate–BSA complex Palmitate–BSA (bovine serum albumin) complex was prepared according to the method described earlier [10]. Briefly, 100 mM sodium palmitate was prepared by incubation of appropriate amount of palmitic acid in 100 mM NaOH at 70 °C for 30 min. Then the solution was gently dropped into triple volume of 10% BSA heated to 52 °C with continuous mixing for 30 min, filtered through 0.45lm syringe filter and stored at 20 °C. Prior to use the solution was heated at 50 °C for 15 min. The actual concentration of palmitate in the complex was determined using NEFA (non-esterified fatty acids) assay kit (Randox). Cells C2C12 mouse myoblasts were grown in DMEM medium supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS) and penicillin plus streptomycin in a humidified atmosphere of 5% CO2/95% air at 37 °C. Cells were split every two days, when their confluence did not exceed 70%. To stimulate cell differentiation cells were grown to 100% confluence and then the regular growth medium was replaced with differentiating medium containing 2% horse serum instead of FBS. Nicorandil (finally 500 nM) or NS1619 (finally 30 nM) dissolved in DMSO was added to the medium 72 h prior to an experiment. Insulin resistance was induced by incubation of cells with 750 lM sodium palmitate (as a complex with BSA) for 24 h prior to experiment. [3H]2-deoxyglucose (2-DOG) uptake 2-DOG uptake was tested according to [11] with small modifications. Briefly, monolayer of myotubes was rinsed with Krebs–Henseleit (KH) buffer and incubated without any exogenous substrates for 30 min. Then, myotubes were stimulated with 10 nM insulin diluted in glucose-free KH buffer for 20 min. [3H] 2-DOG (2 lCi/ml in 1 lM unlabelled 2-DOG, Moravek Biochemicals, Brea, CA, USA) was added and cells were incubated for a next 5 min. Uptake of 2-DOG was terminated by addition of
10 lM cytochalasin B (Sigma–Aldrich Co., St. Louis, MO, USA) for 1 min. Then, the cells were washed three times in ice-cold 0.9% NaCl, solubilized in 0.5 M NaOH and protein concentration was determined. Aliquots of solubilized myotubes were transferred into scintillation vials and diluted with 5 ml of dioxin-based scintillation cocktail. Radioactivity was measured using a liquid scintillation counter (Beckmann). All samples were made in duplicates. The results were corrected for non-specific labeling of cells preincubated with cytochalasin B prior to addition of radioactive 2-DOG. Preparation of cell lysates and Western blotting Myotubes grown in 10-cm tissue culture dishes were rinsed with PBS, trypsinized, centrifuged and rinsed twice with cold (4 °C) PBS (phosphate-buffered saline). Then, 400 ll of ice-cold lysis buffer (Cell Signalling Technology) supplemented with protease inhibitors cocktail (Roche Diagnostic GmbH), NaF (10 mM) and PMSF (1 mM) was added to each sample and the suspension was forced through a thin needle seven times. The lysates were incubated for 20 min on ice and centrifuged at 15 000 g at 4 °C for 20 min. Supernatants were transferred into fresh tubes and protein concentration was measured. An appropriate amount of the sample buffer was added to the lysates and the samples were heated at 95 °C for 5 min. After cooling, they were stored at 20 °C. Polyacrylamide gel electrophoresis (PAGE) was performed under denaturing conditions in the presence of 0.1% sodium dodecyl sulfate. Akt and phospho-Akt (Ser473) protein was detected with the use of specific antibodies (Cell Signalling). All secondary antibodies conjugated with horseradish peroxidase (HRP) were obtained from Abcam. Chemiluminescent substrates Luminata Classic or Crescendo (Millipore) were used for HRP detection. The intensity of bands corresponding to appropriate proteins was calculated densitometrically and expressed in relation to the band corresponding to b-actin (monoclonal anti-b-actin-peroxidaseconjugated antibody, Sigma). Oxygen consumption Cellular oxygen consumption was measured polarographically using OROBOROS Oxygraph-2k (OROBOROSÒ INSTRUMENTS GmbH, Austria). Myotubes grown in 10-cm tissue culture dishes were trypsinized, suspended in BSA-containing growth medium, centrifuged and resuspended in 2 ml of PBS pre-warmed to 37 °C. The protein concentration was approximately 0.5 mg per 1 ml. Oxygen consumption was measured in the presence of sequentially added respiratory substrates and inhibitors (1 mM pyruvate, 5 mM glutamate, 0.1 lg/ml oligomycin, 0.5 lM CCCP) and expressed as pmol of O2 per second per mg of protein. Mitochondrial membrane potential (DW) An effect of cell treatment on DW was estimated fluorimetrically with JC-1 dye (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide; Molecular Probes, Invitrogen) according to the method published by Cossarizza [12]. Myotubes grown in 24-well culture plates were stained with JC-1 dye at 5 lM concentration in the culture medium and incubated for 15 min at 37 °C in the dark. Then, cells were rinsed three times with the culture medium and three times with PBS. Finally, 0.5 ml of PBS was added to each well. For complete DW dissipation, cells were treated with 2 lg/ml of valinomycin plus 5 lM CCCP prior to JC-1 staining. Fluorescence was measured on a laser scanning cytometer iCYS (with excitation at 488 nm). The data was presented as the ratio of orange to green fluorescence representing fully energized and deenergized mitochondria, respectively.
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Reactive oxygen species measurement Reactive oxygen species were measured with carboxy – 20 ,70 difluorodihydrofluorescein diacetate (DFFH2-DA) (Molecular Probes, Invitrogen). Myotubes grown on 12-well plates were stained with 10 lM DFFH2-DA in PBS for 30 min in 37 °C in the dark. Then, fluorescence was measured at 520 nm with excitation at 485 nm on an Infinite 200 micro plate reader (Tecan). Total fluorescence was normalized to the amount of cell protein in each well. Protein measurement Protein concentration was measured using a modified Lowry protein assay kit (Thermo Scientific, Rockford, IL, USA). Statistical analysis Whenever measurement units are meaningless, data are shown as means of ratios of treatment to control values ±SD, for the number of replicates indicated in the figure legends. One-sample t-test was performed on these ratios to test for deviation from unity and associated confidence intervals were computed. Since the normality assumption cannot be strictly met and positive skewness may be expected, analogous computations were repeated for log-transformed values. The resulting p-values and confidence intervals were usually very similar for the both methods of calculation. Other ratios of interest were estimated and are referred to in the same way. To avoid overstating the significance of the outcomes of our experiments we refer to the higher (conservative) p-values and wider confidence limits. Results and discussion Incubation of C2C12 myotubes with insulin results in an increased level of Akt phosphorylation. This is a commonly accepted marker of cellular sensitivity to this hormone. C2C12 myotubes pre-treated with 750 lM palmitate exhibit a substantially reduced Akt phosphorylation upon stimulation with insulin. This wellknown effect reflects fatty acid-induced insulin resistance of muscle cells [13]. As shown in Fig. 1, the latter was efficiently prevented when the C2C12 myotubes were preincubated with nicorandil prior to the addition of palmitate to the growth medium. Nicorandil is an opener of the KATP channel in both the plasma membrane and mitochondria. The mitochondrial component of its action is blocked when nicorandil is applied together with 100 lM 5-HD (5-hydroxydecanoate) which is thought to selectively inhibit KATP in the inner mitochondrial membrane but not in the plasma membrane [14]. As shown in Fig. 1, 5-HD completely abolishes the effect of nicorandil, indicating, that the mitochondrial KATP channel is solely responsible for the nicorandil-dependent prevention of palmitate-induced insulin resistance. It is noteworthy that nicorandil also prevented palmitate-induced insulin resistance in AS-30D hepatoma cells (data not shown), confirming the results for myotubes shown here and indicating some universality of its action. Since phosphorylation of Akt in myotubes treated with insulin precedes the stimulation of glucose uptake from the extracellular space, a protective action of nicorandil on this latter step of cell stimulation should also be expected. Fig. 2 shows that, according to a well-known mechanism, insulin activates [H3]deoxyglucose (DOG) transport into C2C12 cells [15,16], and this response is diminished by palmitate but preserved in cells pretreated with nicorandil. Moreover, nicorandil enhances the response of control myotubes indicating their increased sensitivity to insulin. Opening of the mitochondrial potassium channels is thought to partially dissipate DW, thus one could expect that the effect of nic-
Fig. 1. Effect of nicorandil on Akt phosphorylation in C2C12 myotubes treated with palmitate. (A). Representative immunoblot out of five. (B). Combined data from at least five independent experiments represent mean ± S.D. Myotubes were preincubated with 0.5 lM nicorandil and 750 lM palmitate for 72 and 24 h prior to experiments, respectively. Statistical significance marks for comparisons of interest: stars indicate comparison with control, # indicates comparisons with palmitate alone, @ indicates comparison with palmitate plus nicorandil. ⁄p < 0.001, ⁄⁄ p < 0.005, #p < 0.002, @p < 0.04 Corresponding 95% confidence intervals for ratios are: palmitate vs. control [0.44, 0.66]; nicorandil vs. control [0.84, 1.90]; palmitate + nicorandil vs. palmitate [1.84, 3.56]; palmitate + nicorandil + 5HD vs. control [0.30, 0.70] and palmitate + nicorandil + 5HD vs. palmitate + nicorandil [0.14, 0.74].
orandil on the cellular insulin sensitivity should be correlated with enhanced respiration of the cells. However, the unchanged orange/ green JC-1 fluorescence ratio in cells incubated with nicorandil, relative to that ratio in untreated cells as shown in Fig. 3A, does not indicate any decrease of mitochondrial membrane potential. In addition, only a very slight tendency indicating an increased oxygen consumption was observed (Fig. 3B). This small trend particularly visible after inhibition of ATP-synthase with oligomycin together with a lack of any effect of nicorandil on CCCP-induced oxygen consumption could suggest only minor uncoupling of the oxidative phosphorylation by nicorandil rather than a slight increase of the total mitochondrial respiratory capacity. The latter presumption could stay in line with opening of ATP-activated potassium channels by nicorandil and is consistent with data published elsewhere [17]. This observation may be reasonable in view of the fact that the dependency between changes of the rate of oxygen consumption and the concomitant changes of mitochondrial potential is nonlinear [18], thus the minor (actually almost negligible) stimulation of
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Fig. 2. Effect of nicorandil on deoxyglucose uptake by C2C12 myotubes treated with palmitate. Myotubes were preincubated with 0.5 lM nicorandil and 750 lM palmitate for 72 and 24 h prior to experiments, respectively. Statistical significance marks for comparison with control ⁄p < 0.05 and with palmitate # < 0.05. Corresponding 95% confidence intervals for ratios are: palmitate vs. control [0.71, 0.97]; nicorandil vs. control [1.05, 1.31]; nicorandil + palmitate vs. palmitate [0.95, 1.44]. Number of replicates was 4 for each comparison.
oxygen consumption shown in Fig. 3B can still be compatible with a lack of detectable change of DW as measured with the JC-1 fluorescence method (Fig. 3A), the sensitivity of which is not very high. In fact, Bednarczyk and co-workers [19] have suggested that the dissipation of the DW due to the opening of K+ channels is very low, thus it rather cannot be considered as a mechanism responsible for cytoprotection (see below). On the other hand, a substantial stimulatory effect on oxygen consumption was described for concentrations of nicorandil much higher than those used in the present study [9]. Interestingly, incubation of C2C12 myotubes with nicorandil does not decrease the palmitate-induced ROS generation. In fact, it slightly enhances reactive oxygen species formation in control (insulin non-resistant) cells as well as in those treated with palmitate (Fig. 3C). This phenomenon seems somewhat surprising in view of the cytoprotective action of this compound postulated on the basis of reduced oxidative stress, as it was found earlier [20]. The prevention of insulin resistance by activation of potassium channels is not limited to the KATP channels. A similar effect was observed in C2C12 myotubes preincubated with NS1619, an opener of a large conductance potassium channel activated by Ca2+ and independent of ATP, called BK channel (Fig. 4A). Unfortunately, no selective plasma membrane permeating inhibitor that would allow distinguishing between the mitochondrial BK channels and those located in the plasma membrane is available. Thus, the prevention of the NS1619 effects by paxilline (data not shown), which is a known BK channel blocker, cannot be interpreted unequivocally. Similarly as in the case of nicorandil NS1619 does not affect DW and only very slightly increases oxygen consumption (data not shown) but substantially enhances ROS generation (Fig. 4B). The increased ROS production in myotubes incubated with either nicorandil or NS1619 is in disagreement with the majority of data presented elsewhere. It has convincingly been shown that opening of KATP (with 100 lM nicorandil for 20 min) decreases ROS formation and therefore protects cardiomyocytes from the oxidative damage induced by ischemia–reperfusion [8]. The same was concluded by Jin and co-workers who found protective effects of 30 lM NS1619 or 100 lM diazoxide (another KATP activator) in
Fig. 3. Effect of nicorandil on inner mitochondrial membrane potential, oxygen consumption, and reactive oxygen species generation in C2C12 myotubes. (A). Mitochondrial membrane potential expressed as orange to green fluorescence ratio of JC-1. In control cells it is assumed as 100%. Data represent ± S.D. from five independent experiments. Data for CCCP plus valinomycin are to show relative change of fluorescence upon complete dissipation of the mitochondrial potential. (B). Oxygen consumption by myotubes incubated in the presence of endogenous substrates only followed by sequential addition of pyruvate plus glutamate, oligomycin and mitochondrial uncoupler (CCCP). Oxygen consumption by control cells is assumed as 100%. Collected data represent mean ± S.D. from five independent experiments. (C). Reactive oxygen species formation estimated with the use of DFFH2-DA fluorescent probe. Control value is assumed as 100%. Data represent mean ± S.D. for three independent experiments. ⁄p < 0.002, ⁄⁄<0.006 vs. control cells. Confidence intervals for ratios between values for cells treated with palmitate alone, nicorandil alone and palmitate plus nicorandil and value for control cells assumed as 1 were [1.0590, 1.3954], [1.0207, 1.1705] and [1.1164, 1.5595], respectively.
cardiac cells subjected to ischemia and reperfusion [21]. A similar effect of 100 lM nicorandil was described for isolated muscle mitochondria challenged by ischemia–reperfusion [22]. However, the crucial feature of those earlier experiments differing them from the ones described in this paper is the very high concentration of the potassium channel openers used. In our preliminary experiments incubation of C2C12 myotubes with 30 lM NS1619 for 24 h resulted in substantial cell death. In all experiments shown here NS1619 and nicorandil were at 30 nM and
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Fig. 4. Effect of NS1619 on insulin resistance and reactive oxygen species formation in C2C12 myotubes. Myotubes were preincubated with 30 nM NS1619 and 750 lM palmitate for 72 and 24 h prior to experiments, respectively. (A). Collected data derived from three independent experiments represent mean ± S.D. ⁄p < 0.002, ⁄⁄ p < 0.03, #p < 0.05. ⁄ and ⁄⁄ refer to statistical significance vs. control while # vs. palmitate alone. (B). Reactive oxygen species formation estimated with the use of DFFH2-DA fluorescent probe. Control value is assumed as 100%. Data represent mean ± S.D. for three independent experiments. ⁄p < 0.04, ⁄⁄p < 0.05 vs. control cells. Because of low number of experiments (three) confidence intervals were very wide thus precision of estimations was very low. Data not shown.
500 nM concentration, respectively. It is 2–3 orders of magnitude less than in the majority of electrophysiological studies. Moreover, the stimulation of mitochondrial ROS generation (reduced/prevented by potassium channel openers) described in the papers cited was achieved by temporary cessation of oxygen delivery followed by reperfusion, a procedure that induces additional severe effects on mitochondrial metabolism. The action of the potassium channel openers in such cells cannot be compared directly with their effects in untreated cells or cells treated with palmitate only. Finally, to protect cells against oxidative damage due to acute ischemic insult followed by reperfusion, the potassium channel openers were applied for relatively short period to induce immediate mitochondrial response. In contrast the aim of the experiments shown here was to prevent palmitate-evoked insulin resistance in vitro which is a model of dyslipidaemia-induced weakening of insulin sensitivity in vivo. The latter is a chronic metabolic state, and its therapy needs a prolonged pharmacological treatment. To mimic such a situation both nicorandil and NS 1619 were applied for 72 h (48 h prior to addition of palmitate). Thus, additional longterm effects (but still sensitive to 5-HD in the case of nicorandil use) cannot be excluded. The stimulation of ROS formation in cells treated with KATP openers has also been broadly investigated and discussed. In rabbit cardiomyocytes opening of the mitochondrial KATP by P1075 compound resulted in an increased ROS generation which was sensitive to 5-HD or glibenclamide [23]. Similarly, in vascular smooth muscle diazoxide and pinacidil stimulated ROS generation and this
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effect was efficiently blocked by 5-HD. Valinomycin, which is a potassium-selective ionophore, also increased ROS generation while mitochondrial uncoupling with DNP prevented its effects [24]. This strongly suggests that potassium movement into mitochondria, but not a dissipation of the mitochondrial membrane potential, are necessary for the stimulation of ROS formation. It has been suggested that an enhanced K+ movement to the mitochondrial matrix is counterbalanced by the opposite flux of H+. The resulting alkalinisation of the mitochondrial interior results in an increased ROS generation [25]. In addition, mitochondria are not the only source of ROS in animal cells. Incubation with palmitate results in enhanced ROS generation by NADPH oxidase as described for hepatoma cells [2], cultured hepatocytes [26], and beta cells [27]. In the skeletal muscle both mitochondrial and extramitochondrial palmitate-induced ROS generation has been reported [28]. There are also data indicating that nicorandil, apart from its protective properties which are expressed by limiting of the infarct size in ischemic myocardium [29] may increase ROS generation in isolated cardiomyocytes [30]. Both effects are prevented by 5-HD, suggesting mitochondrial participation. Conversely, the neuroprotective effect of NS1619 has been postulated to be independent of mitochondrial BK channels and attributed to PI3K pathway activation by increased ROS production in cells treated with this drug [31]. An opposite hypothesis of a close link between the BK channels, Ca2+ sensing and cell survival was put forward by Skalska and co-workers [32]. Apart from its action on the BK channel, NS1619 at micromolar concentrations inhibits respiratory chain in glioma cells leading to dissipation of DW independently of potassium channel opening [33]. On the other hand, in isolated brain mitochondria incubated in the presence of calcium ions NS1619 stimulates oxygen consumption even at a concentration as high as 15 lM [32]. A similar conclusion was drawn from experiments with isolated skeletal muscle mitochondria treated with NS1619 at P 1 lM. A cytoprotective effect of NS1619 at a concentrations as high as 30 lM in C2C12 cells challenged with H2O2 was also observed [34]. One may conclude from the above data that the involvement of mitochondrial potassium channels in cytoprotection is multifaceted and their mechanism of action far from being resolved. It seems that the effect of potassium channel openers depends strongly on their concentration and the cell type. It also likely differs when tested on isolated mitochondria and intact cells. In this paper the potassium channel openers were used at the lowest concentrations found to affect cellular insulin resistance. They were much lower than those usually applied for cytoprotection. Using intact cells, which was necessary when the cellular response to insulin was tested, we avoided the obvious drawback of studying isolated mitochondria out of their cellular context. While working with potassium channels activators or blockers one must be aware that pharmacological modulation of potassium fluxes through the plasma membrane and/or the inner mitochondrial membrane in b-cells could potentially have unwanted effects on insulin secretion, as it strongly depends on both the efficiency of the oxidative phosphorylation and the polarization state of the plasma membrane. Such possible consequences must be excluded if a potassium channel opener is considered as a potential drug. We therefore ran an ELISA test for glucose-induced insulin secretion by INS-1E cells treated with nicorandil or NS1619 in a wide range of concentrations, finding no negative effects (data not shown). Also earlier data suggest a lack of any effect of nicorandil on insulin secretion [35–37]. In summary, earlier studies have shown that stimulation of mitochondrial potassium channels promotes cell preconditioning which reduces ischemia–reperfusion-induced damage to cardiomyocytes and may help prevent cardiac complications secondary to ischemic insult [8,21]. Here we show that 5-HD-sensitive
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activation of the mitochondrial potassium channel with nicorandil prevents insulin resistance of myotubes, indicating a novel potential target in the therapy of type 2 diabetes and prediabetic conditions. The similar effects observed with NS1619 suggest common metabolic consequences of activation of KATP and BK channels. However, full understanding of the mechanism linking the activation of mitochondrial potassium channels with improved insulin sensitivity of muscle cells needs further investigation. Acknowledgment This work was supported by the National Center for Research and Development, grant number R13 0043 06/2009. The authors are very grateful to Dr. Tomasz Wyszomirski for his help with statistical analysis. References [1] L. Yuzefovych, G. Wilson, L. Rachek, Endocrinol. Metab. 299 (2010) E1096– E1105. [2] G. Gao, S. Nong, X. Huang, Y. Lu, H. Zhao, Y. Lin, Y. Man, S. Wang, J. Yang, J. Li, J. Biol. Chem. 285 (2010) 29965–29973. [3] S. Nakamura, T. Takamura, N. Matsuzawa-Nagata, H. Takayama, H. Misu, H. Noda, S. Nabemoto, S. Kurita, T. Ota, H. Ando, K. Miyamoto, S. Kaneko, J. Biol. Chem. 284 (2009) 14809–14818. [4] J.A. Maassen, Minerva Med. 99 (2008) 241–251. [5] E.H. Koh, W.J. Lee, Curr. Diabetes Rev. 1 (2005) 331–336. [6] M. Gaster, Biochim. Biophys. Acta 1772 (2007) 755–765. [7] L.C. Martineau, Biochim. Biophys. Acta 1820 (2012) 133–150. [8] R.S. Carreira, P. Monteiro, A.J. Kowaltowski, L.M. Gonçalves, L.A. Providência, J. Bioener, J. Bioener. Biomembr 40 (2008) 95–102. [9] D. Debska, A. Kicinska, J. Skalska, A. Szewczyk, R. May, C.E. Elger, W.S. Kunz, Biochim. Biophys. Acta 1556 (2002) 97–105. [10] S. Cousin, S.R. Hugl, C.E. Wrede, H. Kajio, M.G. Myers, C.J. Rhodes, Endocrinology 142 (2001) 229–240. [11] J.H. Lim, J.I. Lee, Y.H. Suh, W. Kim, J.H. Song, M.H. Jung, Diabetologia 49 (2006) 1924–1936. [12] A. Cossarizza, M. Baccarani-Contri, G. Kalashnikova, C. Franceschi, Biochem. Biophys. Res. Commun. 197 (1993) 40–45. [13] C. Schmitz-Peiffer, D.L. Craig, T.J. Biden, J. Biol. Chem. 274 (1999) 24202– 24210.
[14] S. Vajda, I. Baczkó, I. Leprán, Eur. J. Pharmacol. 577 (2007) 115–123. [15] S.M. Hirabara, R. Curi, P. Maechler, J. Cell. Physiol. 222 (2010) 187–194. [16] X.-T. Feng, T.-Z. Wang, J. Leng, Y. Chen, J.-B. Liu, Biosci. Biotechnol. Biochem. 76 (2012) 1356–1361. [17] A.J. Kowaltowski, S. Seetharaman, P. Paucek, K.D. Garlid, Am. J. Physiol. Circ. Physiol. 280 (2001) H641–H657. [18] D.G. Nicholls, S.J. Ferguson, Bioenergetics 3, third ed., Academic Press, Amsterdam, Boston, London, New York, Oxford, Paris, San Diego, San Francisco, Singapore, Sydney, Tokyo, 2002. [19] P. Bednarczyk, G.D. Barker, A.P. Halestrap, Biochim. Biophys. Acta 1777 (2008) 540–548. [20] I. Khaliulin, H. Schwalb, P. Wang, E. Houminer, L. Grinberg, H. Katzeff, J.B. Borman, S.R. Powell, Biol. Med. 37 (2004) 1–9. [21] C. Jin, J. Wu, M. Watanabe, T. Okada, T. Iesaki, J. Physiol. Sci. 62 (2012) 325– 332. [22] C. Ozcan, M. Bienengraeber, P.P. Dzieja, A. Terzic, Am. J. Physiol. Heart Circ. Physiol. 282 (2002) H531–H539. [23] O. Oldenburg, J. Mol. Cell. Cardiol. 35 (2003) 1035–1042. [24] M. Krenz, O. Oldenburg, H. Wimpee, M.V. Cohen, K.D. Garlid, S.D. Critz, J.M. Gowney, J.N. Benoit, Basic Res. Cardiol. 97 (2002) 365–373. [25] A. Andrukhiv, A.D. Costa, I.C. West, K.D. Garlid, Am. J. Physiol. Heart Circ. Physiol. 291 (2006) H2067–H2074. [26] C.Y. Han, T. Umemoto, M. Omer, L.J. Den Hartigh, T. Chiba, R. LeBoeuf, C.L. Buller, I.R. Sweet, S. Pennathur, J. Biol. Chem. 287 (2012) 10379–10393. [27] M.F. Graciano, L.R. Santos, R. Curi, A.R. Carpinelli, J. Cell Physiol. 226 (2011) 1110–1117. [28] R.H. Lambertucci, S.M. Hirabara, L.D.R. Silveira, A.C. Levada-Pires, R. Curi, T.C. Pithon-Curi, J. Cell Physiol. 216 (2008) 796–804. [29] Y. Dairaku, T. Miura, N. Harada, M. Kimura, T. Okamura, H. Iwamoto, R. Kametani, M. Yamada, Y. Ikeda, M. Iwatate, S. Kawamura, M. Matsuzaki, Circ. 66 (2002) 411–415. [30] A. Kuno, S.D. Critz, M.V. Cohen, J.M. Downey, Basic Res. Cardiol. 102 (2007) 73– 79. [31] T. Gáspár, P. Katakam, J.A. Snipes, B. Kis, F. Domoki, F. Bari, D.W. Busija, J. Neurochem. 105 (2008) 1115–1128. [32] J. Skalska, P. Bednarczyk, M. Piwon´ska, B. Kulawiak, G. Wilczynski, K. Dołowy, A.P. Kudin, W.S. Kunz, A. Szewczyk, Int. J. Mol. Sci. 10 (2009) 1104–1120. [33] G. Debska, A. Kicinska, J. Dobrucki, B. Dworakowska, E. Nurowska, J. Skalska, K. Dolowy, A. Szewczyk, Biochem. Pharm. 65 (2003) 1827–1834. [34] J. Skalska, M. Piwon´ska, E. Wyroba, L. Surmacz, R. Wieczorek, I. KoszelaPiotrowska, J. Zielin´ska, P. Bednarczyk, K. Dołowy, G.M. Wilczynski, A. Szewczyk, W.S. Kunz, Biochim. Biophys. Acta 1777 (2008) 651–659. [35] F.M. Gribble, F. Reimann, Biochem. Soc. Trans. 30 (2002) 333–339. [36] D.J. Patel, H.J. Purcell, K.M. Fox, Eur. Heart J. 20 (1999) 51–57. [37] T. Wasada, C. Watanabe, T. Nakagami, Y. Iwamoto, Metabolism 48 (1999) 432– 436.
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Potassium channel openers prevent palmitate-induced insulin resistance in C2C12 myotubes Dorota Dymkowska, Beata Drabarek, Justyna Jakubczyk, Sylwia Wojciechowska, Krzysztof Zabłocki ⇑ Nencki Institute of Experimental Biology, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 9 July 2013 and in revised form 28 October 2013 Available online 19 November 2013 Keywords: C2C12 myotubes Insulin resistance Nicorandil NS1619 Reactive oxygen species
a b s t r a c t Insulin resistance (IR) of muscle cells is an early symptom of type 2 diabetes. It often results from excessive lipid accumulation in muscle fibers which under in vitro experimental conditions may be induced by incubation of muscle cells with palmitate. IR is manifested as a reduced response of cells to insulin expressed by lowered Akt kinase phosphorylation and decreased insulin-dependent glucose uptake. Stimulation of mitochondrial oxidative metabolism by mild dissipation of the mitochondrial potential is thought to increase fatty acid utilization and thereby prevent insulin resistance. Here it is shown that nicorandil and NS1619, which are openers of two different mitochondrial potassium channels, protect C2C12 myotubes from palmitate-induced insulin resistance. Preincubation of myotubes with 5-hydroxydecanoate abolishes the protective effect of nicorandil. The efficient concentrations of both openers are far below those commonly applied for cytoprotection. This is probably why their effects on the mitochondrial energy metabolism are small. These data suggest that opening of mitochondrial potassium channels could be a promising approach in prevention and therapy of insulin resistance related to dyslipidemia and obesity. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Insulin resistance is an early symptom of type 2 diabetes, which may appear in apparently healthy young people. It gradually leads to increased blood glucose concentration, glucose intolerance, hyperinsulinemia and finally an impairment of the b-cell function. In many cases IR1 develops in obese individuals, and results from insufficient oxidation of fatty acids in muscle and liver cells. Accumulation of acylglycerol deposits in cells causes an excessive activation of protein kinase C (PKC) and multiple phosphorylation of insulin receptor substrate-1 (IRS-1) at serine residues that prevents its proper phosphorylation catalyzed by the intrinsic insulin receptor tyrosine kinase. In addition, the excessive fatty acid accumulation enhances insulin resistance via several intermediates such as acylCoA, diacylglycerol and ceramides affecting the insulin signaling pathway and glucose metabolism. Moreover, fatty-acid induced oxidative stress is suggested to be an important factor de-sensitizing cells to insulin [1]. The excessive generation of reactive oxygen spe⇑ Corresponding author. Address: Nencki Institute of Experimental Biology, Pasteura str, 02-093 Warsaw, Poland. E-mail address: [email protected] (K. Zabłocki). 1 Abbreviations used: IR, Insulin resistance; PKC, protein kinase C; IRS-1, insulin receptor substrate-1; ROS, reactive oxygen species; NEFA, non-esterified fatty acids; FBS, fetal bovine serum; PAGE, Polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; DFFH 2 -DA, difluorodihydrofluorescein diacetate; 5-HD, 5hydroxydecanoate. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.11.003
cies (ROS) is mostly attributed to the NADPH oxidase activity and the mitochondrial electron transport chain, but their relative contribution to the cellular oxidative stress is still under debate [2,3]. Nevertheless, the link between IR and mitochondrial dysfunction is widely accepted. It has been well documented that stimulation of fatty acid oxidation in the liver and muscle substantially improves the insulin sensitivity of these tissues [4,5]. In line with that observation, it has also been shown that dissipation of mitochondrial membrane potential (DW with mitochondrial uncouplers stimulates fatty acid oxidation and potentiates insulin-stimulated glucose uptake by human cultured myotubes, indicating enhanced insulin sensitivity of these cells [6]. Conversely, inhibition of the respiratory chain with oligomycin and/or antimycin reduces fatty acid oxidation and decreases the insulin sensitivity of myotubes. Mitochondrial uncoupling not only stimulates fatty acid oxidation (restoring insulin sensitivity) but also reduces the cellular energy charge, activating AMPK. Eventually it enhances muscle glucose uptake and limits hepatic gluconeogenesis [7]. Nicorandil is a nitrate derivative of nicotinamide, thus it exerts vasodilatory effects due to vascular smooth muscle relaxation. In addition, it was found to activate the ATP-sensitive K+ channel in cardiac muscle mitochondria. The latter effect is interpreted as a mechanism of pharmacologically induced heart preconditioning based on K+ cycling across the inner mitochondrial membrane and mild mitochondrial membrane depolarization. It prevents Ca2+ overloading of mitochondria and protects cells against
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ischemia-induced injury [8]. The decrease in the mitochondrial membrane potential resulting from the enhanced K+ permeability of the inner mitochondrial membrane correlates with an accelerated oxygen consumption by muscle cells or isolated mitochondria treated with nicorandil [9]. Therefore, the stimulation of respiration following pharmacological opening of mitochondrial potassium channels could be a remedy for insulin resistance, restoring the proper cellular response to this hormone. Data shown here indicate that two potassium channel openers of different specificity (nicorandil and NS1619 activating an ATPsensitive channel, KATP, and the calcium-dependent large-conductance BK channel, respectively) prevent palmitate-induced insulin resistance in C2C12 myotubes. This finding confirms the assumption presented above and indicates that potassium channel openers may be useful in antidiabetic therapy. Furthermore, only mild changes in the mitochondrial metabolism induced by these compounds suggest that even a marginal increase of the mitochondrial inner membrane permeability for potassium is sufficient to prevent insulin resistance. This latter finding together with only slightly increased ROS generation indicate that the mechanism involving potassium channel openers in the prevention of palmitate-induced IR of myotubes is not straightforward. Probably it is not a simple consequence of mitochondrial malfunctioning, but relies on a complex, as yet unidentified regulatory processes. Material and methods Preparation of palmitate–BSA complex Palmitate–BSA (bovine serum albumin) complex was prepared according to the method described earlier [10]. Briefly, 100 mM sodium palmitate was prepared by incubation of appropriate amount of palmitic acid in 100 mM NaOH at 70 °C for 30 min. Then the solution was gently dropped into triple volume of 10% BSA heated to 52 °C with continuous mixing for 30 min, filtered through 0.45lm syringe filter and stored at 20 °C. Prior to use the solution was heated at 50 °C for 15 min. The actual concentration of palmitate in the complex was determined using NEFA (non-esterified fatty acids) assay kit (Randox). Cells C2C12 mouse myoblasts were grown in DMEM medium supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS) and penicillin plus streptomycin in a humidified atmosphere of 5% CO2/95% air at 37 °C. Cells were split every two days, when their confluence did not exceed 70%. To stimulate cell differentiation cells were grown to 100% confluence and then the regular growth medium was replaced with differentiating medium containing 2% horse serum instead of FBS. Nicorandil (finally 500 nM) or NS1619 (finally 30 nM) dissolved in DMSO was added to the medium 72 h prior to an experiment. Insulin resistance was induced by incubation of cells with 750 lM sodium palmitate (as a complex with BSA) for 24 h prior to experiment. [3H]2-deoxyglucose (2-DOG) uptake 2-DOG uptake was tested according to [11] with small modifications. Briefly, monolayer of myotubes was rinsed with Krebs–Henseleit (KH) buffer and incubated without any exogenous substrates for 30 min. Then, myotubes were stimulated with 10 nM insulin diluted in glucose-free KH buffer for 20 min. [3H] 2-DOG (2 lCi/ml in 1 lM unlabelled 2-DOG, Moravek Biochemicals, Brea, CA, USA) was added and cells were incubated for a next 5 min. Uptake of 2-DOG was terminated by addition of
10 lM cytochalasin B (Sigma–Aldrich Co., St. Louis, MO, USA) for 1 min. Then, the cells were washed three times in ice-cold 0.9% NaCl, solubilized in 0.5 M NaOH and protein concentration was determined. Aliquots of solubilized myotubes were transferred into scintillation vials and diluted with 5 ml of dioxin-based scintillation cocktail. Radioactivity was measured using a liquid scintillation counter (Beckmann). All samples were made in duplicates. The results were corrected for non-specific labeling of cells preincubated with cytochalasin B prior to addition of radioactive 2-DOG. Preparation of cell lysates and Western blotting Myotubes grown in 10-cm tissue culture dishes were rinsed with PBS, trypsinized, centrifuged and rinsed twice with cold (4 °C) PBS (phosphate-buffered saline). Then, 400 ll of ice-cold lysis buffer (Cell Signalling Technology) supplemented with protease inhibitors cocktail (Roche Diagnostic GmbH), NaF (10 mM) and PMSF (1 mM) was added to each sample and the suspension was forced through a thin needle seven times. The lysates were incubated for 20 min on ice and centrifuged at 15 000 g at 4 °C for 20 min. Supernatants were transferred into fresh tubes and protein concentration was measured. An appropriate amount of the sample buffer was added to the lysates and the samples were heated at 95 °C for 5 min. After cooling, they were stored at 20 °C. Polyacrylamide gel electrophoresis (PAGE) was performed under denaturing conditions in the presence of 0.1% sodium dodecyl sulfate. Akt and phospho-Akt (Ser473) protein was detected with the use of specific antibodies (Cell Signalling). All secondary antibodies conjugated with horseradish peroxidase (HRP) were obtained from Abcam. Chemiluminescent substrates Luminata Classic or Crescendo (Millipore) were used for HRP detection. The intensity of bands corresponding to appropriate proteins was calculated densitometrically and expressed in relation to the band corresponding to b-actin (monoclonal anti-b-actin-peroxidaseconjugated antibody, Sigma). Oxygen consumption Cellular oxygen consumption was measured polarographically using OROBOROS Oxygraph-2k (OROBOROSÒ INSTRUMENTS GmbH, Austria). Myotubes grown in 10-cm tissue culture dishes were trypsinized, suspended in BSA-containing growth medium, centrifuged and resuspended in 2 ml of PBS pre-warmed to 37 °C. The protein concentration was approximately 0.5 mg per 1 ml. Oxygen consumption was measured in the presence of sequentially added respiratory substrates and inhibitors (1 mM pyruvate, 5 mM glutamate, 0.1 lg/ml oligomycin, 0.5 lM CCCP) and expressed as pmol of O2 per second per mg of protein. Mitochondrial membrane potential (DW) An effect of cell treatment on DW was estimated fluorimetrically with JC-1 dye (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide; Molecular Probes, Invitrogen) according to the method published by Cossarizza [12]. Myotubes grown in 24-well culture plates were stained with JC-1 dye at 5 lM concentration in the culture medium and incubated for 15 min at 37 °C in the dark. Then, cells were rinsed three times with the culture medium and three times with PBS. Finally, 0.5 ml of PBS was added to each well. For complete DW dissipation, cells were treated with 2 lg/ml of valinomycin plus 5 lM CCCP prior to JC-1 staining. Fluorescence was measured on a laser scanning cytometer iCYS (with excitation at 488 nm). The data was presented as the ratio of orange to green fluorescence representing fully energized and deenergized mitochondria, respectively.
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Reactive oxygen species measurement Reactive oxygen species were measured with carboxy – 20 ,70 difluorodihydrofluorescein diacetate (DFFH2-DA) (Molecular Probes, Invitrogen). Myotubes grown on 12-well plates were stained with 10 lM DFFH2-DA in PBS for 30 min in 37 °C in the dark. Then, fluorescence was measured at 520 nm with excitation at 485 nm on an Infinite 200 micro plate reader (Tecan). Total fluorescence was normalized to the amount of cell protein in each well. Protein measurement Protein concentration was measured using a modified Lowry protein assay kit (Thermo Scientific, Rockford, IL, USA). Statistical analysis Whenever measurement units are meaningless, data are shown as means of ratios of treatment to control values ±SD, for the number of replicates indicated in the figure legends. One-sample t-test was performed on these ratios to test for deviation from unity and associated confidence intervals were computed. Since the normality assumption cannot be strictly met and positive skewness may be expected, analogous computations were repeated for log-transformed values. The resulting p-values and confidence intervals were usually very similar for the both methods of calculation. Other ratios of interest were estimated and are referred to in the same way. To avoid overstating the significance of the outcomes of our experiments we refer to the higher (conservative) p-values and wider confidence limits. Results and discussion Incubation of C2C12 myotubes with insulin results in an increased level of Akt phosphorylation. This is a commonly accepted marker of cellular sensitivity to this hormone. C2C12 myotubes pre-treated with 750 lM palmitate exhibit a substantially reduced Akt phosphorylation upon stimulation with insulin. This wellknown effect reflects fatty acid-induced insulin resistance of muscle cells [13]. As shown in Fig. 1, the latter was efficiently prevented when the C2C12 myotubes were preincubated with nicorandil prior to the addition of palmitate to the growth medium. Nicorandil is an opener of the KATP channel in both the plasma membrane and mitochondria. The mitochondrial component of its action is blocked when nicorandil is applied together with 100 lM 5-HD (5-hydroxydecanoate) which is thought to selectively inhibit KATP in the inner mitochondrial membrane but not in the plasma membrane [14]. As shown in Fig. 1, 5-HD completely abolishes the effect of nicorandil, indicating, that the mitochondrial KATP channel is solely responsible for the nicorandil-dependent prevention of palmitate-induced insulin resistance. It is noteworthy that nicorandil also prevented palmitate-induced insulin resistance in AS-30D hepatoma cells (data not shown), confirming the results for myotubes shown here and indicating some universality of its action. Since phosphorylation of Akt in myotubes treated with insulin precedes the stimulation of glucose uptake from the extracellular space, a protective action of nicorandil on this latter step of cell stimulation should also be expected. Fig. 2 shows that, according to a well-known mechanism, insulin activates [H3]deoxyglucose (DOG) transport into C2C12 cells [15,16], and this response is diminished by palmitate but preserved in cells pretreated with nicorandil. Moreover, nicorandil enhances the response of control myotubes indicating their increased sensitivity to insulin. Opening of the mitochondrial potassium channels is thought to partially dissipate DW, thus one could expect that the effect of nic-
Fig. 1. Effect of nicorandil on Akt phosphorylation in C2C12 myotubes treated with palmitate. (A). Representative immunoblot out of five. (B). Combined data from at least five independent experiments represent mean ± S.D. Myotubes were preincubated with 0.5 lM nicorandil and 750 lM palmitate for 72 and 24 h prior to experiments, respectively. Statistical significance marks for comparisons of interest: stars indicate comparison with control, # indicates comparisons with palmitate alone, @ indicates comparison with palmitate plus nicorandil. ⁄p < 0.001, ⁄⁄ p < 0.005, #p < 0.002, @p < 0.04 Corresponding 95% confidence intervals for ratios are: palmitate vs. control [0.44, 0.66]; nicorandil vs. control [0.84, 1.90]; palmitate + nicorandil vs. palmitate [1.84, 3.56]; palmitate + nicorandil + 5HD vs. control [0.30, 0.70] and palmitate + nicorandil + 5HD vs. palmitate + nicorandil [0.14, 0.74].
orandil on the cellular insulin sensitivity should be correlated with enhanced respiration of the cells. However, the unchanged orange/ green JC-1 fluorescence ratio in cells incubated with nicorandil, relative to that ratio in untreated cells as shown in Fig. 3A, does not indicate any decrease of mitochondrial membrane potential. In addition, only a very slight tendency indicating an increased oxygen consumption was observed (Fig. 3B). This small trend particularly visible after inhibition of ATP-synthase with oligomycin together with a lack of any effect of nicorandil on CCCP-induced oxygen consumption could suggest only minor uncoupling of the oxidative phosphorylation by nicorandil rather than a slight increase of the total mitochondrial respiratory capacity. The latter presumption could stay in line with opening of ATP-activated potassium channels by nicorandil and is consistent with data published elsewhere [17]. This observation may be reasonable in view of the fact that the dependency between changes of the rate of oxygen consumption and the concomitant changes of mitochondrial potential is nonlinear [18], thus the minor (actually almost negligible) stimulation of
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Fig. 2. Effect of nicorandil on deoxyglucose uptake by C2C12 myotubes treated with palmitate. Myotubes were preincubated with 0.5 lM nicorandil and 750 lM palmitate for 72 and 24 h prior to experiments, respectively. Statistical significance marks for comparison with control ⁄p < 0.05 and with palmitate # < 0.05. Corresponding 95% confidence intervals for ratios are: palmitate vs. control [0.71, 0.97]; nicorandil vs. control [1.05, 1.31]; nicorandil + palmitate vs. palmitate [0.95, 1.44]. Number of replicates was 4 for each comparison.
oxygen consumption shown in Fig. 3B can still be compatible with a lack of detectable change of DW as measured with the JC-1 fluorescence method (Fig. 3A), the sensitivity of which is not very high. In fact, Bednarczyk and co-workers [19] have suggested that the dissipation of the DW due to the opening of K+ channels is very low, thus it rather cannot be considered as a mechanism responsible for cytoprotection (see below). On the other hand, a substantial stimulatory effect on oxygen consumption was described for concentrations of nicorandil much higher than those used in the present study [9]. Interestingly, incubation of C2C12 myotubes with nicorandil does not decrease the palmitate-induced ROS generation. In fact, it slightly enhances reactive oxygen species formation in control (insulin non-resistant) cells as well as in those treated with palmitate (Fig. 3C). This phenomenon seems somewhat surprising in view of the cytoprotective action of this compound postulated on the basis of reduced oxidative stress, as it was found earlier [20]. The prevention of insulin resistance by activation of potassium channels is not limited to the KATP channels. A similar effect was observed in C2C12 myotubes preincubated with NS1619, an opener of a large conductance potassium channel activated by Ca2+ and independent of ATP, called BK channel (Fig. 4A). Unfortunately, no selective plasma membrane permeating inhibitor that would allow distinguishing between the mitochondrial BK channels and those located in the plasma membrane is available. Thus, the prevention of the NS1619 effects by paxilline (data not shown), which is a known BK channel blocker, cannot be interpreted unequivocally. Similarly as in the case of nicorandil NS1619 does not affect DW and only very slightly increases oxygen consumption (data not shown) but substantially enhances ROS generation (Fig. 4B). The increased ROS production in myotubes incubated with either nicorandil or NS1619 is in disagreement with the majority of data presented elsewhere. It has convincingly been shown that opening of KATP (with 100 lM nicorandil for 20 min) decreases ROS formation and therefore protects cardiomyocytes from the oxidative damage induced by ischemia–reperfusion [8]. The same was concluded by Jin and co-workers who found protective effects of 30 lM NS1619 or 100 lM diazoxide (another KATP activator) in
Fig. 3. Effect of nicorandil on inner mitochondrial membrane potential, oxygen consumption, and reactive oxygen species generation in C2C12 myotubes. (A). Mitochondrial membrane potential expressed as orange to green fluorescence ratio of JC-1. In control cells it is assumed as 100%. Data represent ± S.D. from five independent experiments. Data for CCCP plus valinomycin are to show relative change of fluorescence upon complete dissipation of the mitochondrial potential. (B). Oxygen consumption by myotubes incubated in the presence of endogenous substrates only followed by sequential addition of pyruvate plus glutamate, oligomycin and mitochondrial uncoupler (CCCP). Oxygen consumption by control cells is assumed as 100%. Collected data represent mean ± S.D. from five independent experiments. (C). Reactive oxygen species formation estimated with the use of DFFH2-DA fluorescent probe. Control value is assumed as 100%. Data represent mean ± S.D. for three independent experiments. ⁄p < 0.002, ⁄⁄<0.006 vs. control cells. Confidence intervals for ratios between values for cells treated with palmitate alone, nicorandil alone and palmitate plus nicorandil and value for control cells assumed as 1 were [1.0590, 1.3954], [1.0207, 1.1705] and [1.1164, 1.5595], respectively.
cardiac cells subjected to ischemia and reperfusion [21]. A similar effect of 100 lM nicorandil was described for isolated muscle mitochondria challenged by ischemia–reperfusion [22]. However, the crucial feature of those earlier experiments differing them from the ones described in this paper is the very high concentration of the potassium channel openers used. In our preliminary experiments incubation of C2C12 myotubes with 30 lM NS1619 for 24 h resulted in substantial cell death. In all experiments shown here NS1619 and nicorandil were at 30 nM and
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Fig. 4. Effect of NS1619 on insulin resistance and reactive oxygen species formation in C2C12 myotubes. Myotubes were preincubated with 30 nM NS1619 and 750 lM palmitate for 72 and 24 h prior to experiments, respectively. (A). Collected data derived from three independent experiments represent mean ± S.D. ⁄p < 0.002, ⁄⁄ p < 0.03, #p < 0.05. ⁄ and ⁄⁄ refer to statistical significance vs. control while # vs. palmitate alone. (B). Reactive oxygen species formation estimated with the use of DFFH2-DA fluorescent probe. Control value is assumed as 100%. Data represent mean ± S.D. for three independent experiments. ⁄p < 0.04, ⁄⁄p < 0.05 vs. control cells. Because of low number of experiments (three) confidence intervals were very wide thus precision of estimations was very low. Data not shown.
500 nM concentration, respectively. It is 2–3 orders of magnitude less than in the majority of electrophysiological studies. Moreover, the stimulation of mitochondrial ROS generation (reduced/prevented by potassium channel openers) described in the papers cited was achieved by temporary cessation of oxygen delivery followed by reperfusion, a procedure that induces additional severe effects on mitochondrial metabolism. The action of the potassium channel openers in such cells cannot be compared directly with their effects in untreated cells or cells treated with palmitate only. Finally, to protect cells against oxidative damage due to acute ischemic insult followed by reperfusion, the potassium channel openers were applied for relatively short period to induce immediate mitochondrial response. In contrast the aim of the experiments shown here was to prevent palmitate-evoked insulin resistance in vitro which is a model of dyslipidaemia-induced weakening of insulin sensitivity in vivo. The latter is a chronic metabolic state, and its therapy needs a prolonged pharmacological treatment. To mimic such a situation both nicorandil and NS 1619 were applied for 72 h (48 h prior to addition of palmitate). Thus, additional longterm effects (but still sensitive to 5-HD in the case of nicorandil use) cannot be excluded. The stimulation of ROS formation in cells treated with KATP openers has also been broadly investigated and discussed. In rabbit cardiomyocytes opening of the mitochondrial KATP by P1075 compound resulted in an increased ROS generation which was sensitive to 5-HD or glibenclamide [23]. Similarly, in vascular smooth muscle diazoxide and pinacidil stimulated ROS generation and this
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effect was efficiently blocked by 5-HD. Valinomycin, which is a potassium-selective ionophore, also increased ROS generation while mitochondrial uncoupling with DNP prevented its effects [24]. This strongly suggests that potassium movement into mitochondria, but not a dissipation of the mitochondrial membrane potential, are necessary for the stimulation of ROS formation. It has been suggested that an enhanced K+ movement to the mitochondrial matrix is counterbalanced by the opposite flux of H+. The resulting alkalinisation of the mitochondrial interior results in an increased ROS generation [25]. In addition, mitochondria are not the only source of ROS in animal cells. Incubation with palmitate results in enhanced ROS generation by NADPH oxidase as described for hepatoma cells [2], cultured hepatocytes [26], and beta cells [27]. In the skeletal muscle both mitochondrial and extramitochondrial palmitate-induced ROS generation has been reported [28]. There are also data indicating that nicorandil, apart from its protective properties which are expressed by limiting of the infarct size in ischemic myocardium [29] may increase ROS generation in isolated cardiomyocytes [30]. Both effects are prevented by 5-HD, suggesting mitochondrial participation. Conversely, the neuroprotective effect of NS1619 has been postulated to be independent of mitochondrial BK channels and attributed to PI3K pathway activation by increased ROS production in cells treated with this drug [31]. An opposite hypothesis of a close link between the BK channels, Ca2+ sensing and cell survival was put forward by Skalska and co-workers [32]. Apart from its action on the BK channel, NS1619 at micromolar concentrations inhibits respiratory chain in glioma cells leading to dissipation of DW independently of potassium channel opening [33]. On the other hand, in isolated brain mitochondria incubated in the presence of calcium ions NS1619 stimulates oxygen consumption even at a concentration as high as 15 lM [32]. A similar conclusion was drawn from experiments with isolated skeletal muscle mitochondria treated with NS1619 at P 1 lM. A cytoprotective effect of NS1619 at a concentrations as high as 30 lM in C2C12 cells challenged with H2O2 was also observed [34]. One may conclude from the above data that the involvement of mitochondrial potassium channels in cytoprotection is multifaceted and their mechanism of action far from being resolved. It seems that the effect of potassium channel openers depends strongly on their concentration and the cell type. It also likely differs when tested on isolated mitochondria and intact cells. In this paper the potassium channel openers were used at the lowest concentrations found to affect cellular insulin resistance. They were much lower than those usually applied for cytoprotection. Using intact cells, which was necessary when the cellular response to insulin was tested, we avoided the obvious drawback of studying isolated mitochondria out of their cellular context. While working with potassium channels activators or blockers one must be aware that pharmacological modulation of potassium fluxes through the plasma membrane and/or the inner mitochondrial membrane in b-cells could potentially have unwanted effects on insulin secretion, as it strongly depends on both the efficiency of the oxidative phosphorylation and the polarization state of the plasma membrane. Such possible consequences must be excluded if a potassium channel opener is considered as a potential drug. We therefore ran an ELISA test for glucose-induced insulin secretion by INS-1E cells treated with nicorandil or NS1619 in a wide range of concentrations, finding no negative effects (data not shown). Also earlier data suggest a lack of any effect of nicorandil on insulin secretion [35–37]. In summary, earlier studies have shown that stimulation of mitochondrial potassium channels promotes cell preconditioning which reduces ischemia–reperfusion-induced damage to cardiomyocytes and may help prevent cardiac complications secondary to ischemic insult [8,21]. Here we show that 5-HD-sensitive
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activation of the mitochondrial potassium channel with nicorandil prevents insulin resistance of myotubes, indicating a novel potential target in the therapy of type 2 diabetes and prediabetic conditions. The similar effects observed with NS1619 suggest common metabolic consequences of activation of KATP and BK channels. However, full understanding of the mechanism linking the activation of mitochondrial potassium channels with improved insulin sensitivity of muscle cells needs further investigation. Acknowledgment This work was supported by the National Center for Research and Development, grant number R13 0043 06/2009. The authors are very grateful to Dr. Tomasz Wyszomirski for his help with statistical analysis. References [1] L. Yuzefovych, G. Wilson, L. Rachek, Endocrinol. Metab. 299 (2010) E1096– E1105. [2] G. Gao, S. Nong, X. Huang, Y. Lu, H. Zhao, Y. Lin, Y. Man, S. Wang, J. Yang, J. Li, J. Biol. Chem. 285 (2010) 29965–29973. [3] S. Nakamura, T. Takamura, N. Matsuzawa-Nagata, H. Takayama, H. Misu, H. Noda, S. Nabemoto, S. Kurita, T. Ota, H. Ando, K. Miyamoto, S. Kaneko, J. Biol. Chem. 284 (2009) 14809–14818. [4] J.A. Maassen, Minerva Med. 99 (2008) 241–251. [5] E.H. Koh, W.J. Lee, Curr. Diabetes Rev. 1 (2005) 331–336. [6] M. Gaster, Biochim. Biophys. Acta 1772 (2007) 755–765. [7] L.C. Martineau, Biochim. Biophys. Acta 1820 (2012) 133–150. [8] R.S. Carreira, P. Monteiro, A.J. Kowaltowski, L.M. Gonçalves, L.A. Providência, J. Bioener, J. Bioener. Biomembr 40 (2008) 95–102. [9] D. Debska, A. Kicinska, J. Skalska, A. Szewczyk, R. May, C.E. Elger, W.S. Kunz, Biochim. Biophys. Acta 1556 (2002) 97–105. [10] S. Cousin, S.R. Hugl, C.E. Wrede, H. Kajio, M.G. Myers, C.J. Rhodes, Endocrinology 142 (2001) 229–240. [11] J.H. Lim, J.I. Lee, Y.H. Suh, W. Kim, J.H. Song, M.H. Jung, Diabetologia 49 (2006) 1924–1936. [12] A. Cossarizza, M. Baccarani-Contri, G. Kalashnikova, C. Franceschi, Biochem. Biophys. Res. Commun. 197 (1993) 40–45. [13] C. Schmitz-Peiffer, D.L. Craig, T.J. Biden, J. Biol. Chem. 274 (1999) 24202– 24210.
[14] S. Vajda, I. Baczkó, I. Leprán, Eur. J. Pharmacol. 577 (2007) 115–123. [15] S.M. Hirabara, R. Curi, P. Maechler, J. Cell. Physiol. 222 (2010) 187–194. [16] X.-T. Feng, T.-Z. Wang, J. Leng, Y. Chen, J.-B. Liu, Biosci. Biotechnol. Biochem. 76 (2012) 1356–1361. [17] A.J. Kowaltowski, S. Seetharaman, P. Paucek, K.D. Garlid, Am. J. Physiol. Circ. Physiol. 280 (2001) H641–H657. [18] D.G. Nicholls, S.J. Ferguson, Bioenergetics 3, third ed., Academic Press, Amsterdam, Boston, London, New York, Oxford, Paris, San Diego, San Francisco, Singapore, Sydney, Tokyo, 2002. [19] P. Bednarczyk, G.D. Barker, A.P. Halestrap, Biochim. Biophys. Acta 1777 (2008) 540–548. [20] I. Khaliulin, H. Schwalb, P. Wang, E. Houminer, L. Grinberg, H. Katzeff, J.B. Borman, S.R. Powell, Biol. Med. 37 (2004) 1–9. [21] C. Jin, J. Wu, M. Watanabe, T. Okada, T. Iesaki, J. Physiol. Sci. 62 (2012) 325– 332. [22] C. Ozcan, M. Bienengraeber, P.P. Dzieja, A. Terzic, Am. J. Physiol. Heart Circ. Physiol. 282 (2002) H531–H539. [23] O. Oldenburg, J. Mol. Cell. Cardiol. 35 (2003) 1035–1042. [24] M. Krenz, O. Oldenburg, H. Wimpee, M.V. Cohen, K.D. Garlid, S.D. Critz, J.M. Gowney, J.N. Benoit, Basic Res. Cardiol. 97 (2002) 365–373. [25] A. Andrukhiv, A.D. Costa, I.C. West, K.D. Garlid, Am. J. Physiol. Heart Circ. Physiol. 291 (2006) H2067–H2074. [26] C.Y. Han, T. Umemoto, M. Omer, L.J. Den Hartigh, T. Chiba, R. LeBoeuf, C.L. Buller, I.R. Sweet, S. Pennathur, J. Biol. Chem. 287 (2012) 10379–10393. [27] M.F. Graciano, L.R. Santos, R. Curi, A.R. Carpinelli, J. Cell Physiol. 226 (2011) 1110–1117. [28] R.H. Lambertucci, S.M. Hirabara, L.D.R. Silveira, A.C. Levada-Pires, R. Curi, T.C. Pithon-Curi, J. Cell Physiol. 216 (2008) 796–804. [29] Y. Dairaku, T. Miura, N. Harada, M. Kimura, T. Okamura, H. Iwamoto, R. Kametani, M. Yamada, Y. Ikeda, M. Iwatate, S. Kawamura, M. Matsuzaki, Circ. 66 (2002) 411–415. [30] A. Kuno, S.D. Critz, M.V. Cohen, J.M. Downey, Basic Res. Cardiol. 102 (2007) 73– 79. [31] T. Gáspár, P. Katakam, J.A. Snipes, B. Kis, F. Domoki, F. Bari, D.W. Busija, J. Neurochem. 105 (2008) 1115–1128. [32] J. Skalska, P. Bednarczyk, M. Piwon´ska, B. Kulawiak, G. Wilczynski, K. Dołowy, A.P. Kudin, W.S. Kunz, A. Szewczyk, Int. J. Mol. Sci. 10 (2009) 1104–1120. [33] G. Debska, A. Kicinska, J. Dobrucki, B. Dworakowska, E. Nurowska, J. Skalska, K. Dolowy, A. Szewczyk, Biochem. Pharm. 65 (2003) 1827–1834. [34] J. Skalska, M. Piwon´ska, E. Wyroba, L. Surmacz, R. Wieczorek, I. KoszelaPiotrowska, J. Zielin´ska, P. Bednarczyk, K. Dołowy, G.M. Wilczynski, A. Szewczyk, W.S. Kunz, Biochim. Biophys. Acta 1777 (2008) 651–659. [35] F.M. Gribble, F. Reimann, Biochem. Soc. Trans. 30 (2002) 333–339. [36] D.J. Patel, H.J. Purcell, K.M. Fox, Eur. Heart J. 20 (1999) 51–57. [37] T. Wasada, C. Watanabe, T. Nakagami, Y. Iwamoto, Metabolism 48 (1999) 432– 436.