Introduction

Activation of opioid receptors induces various effects [1], including pain control [2], the modulation of immune function [3] and the regulation of glucose metabolism [4]. Chemical agents such as loperamide and tramadol decreased plasma glucose via activation of opioid μ-receptors in a type 1 diabetes rat model [5, 6]. Insulin resistance is described as the reduced response of peripheral tissues to insulin [7]. Infusion of β-endorphin may improve insulin resistance in fructose-rich chow-fed rats [8]. Indeed, the induction of insulin resistance was more rapid in opioid μ-receptor knock-out mice than in wild-type mice [9]. Activation of the opioid μ-receptor to improve insulin resistance seems plausible.

Insulin resistance is related to an increase of inflammatory markers including TNF-α and IL-6 in the acute phase [10, 11]. The correlation between IL-6 concentration and the severity of insulin resistance in patients was higher than that of TNF-α [12]. Similar to adipose tissue, skeletal muscle is the major tissue of insulin-stimulated glucose disposal [13]. Insulin increases IL-6 gene expression and circulating IL-6 level in patients with insulin resistance, but not in patients with healthy skeletal muscle; IL-6 is important for the induction of insulin resistance [14]. Mouse C2C12 cells derived from the mouse skeletal muscle C2 cell line express the similar properties of isolated skeletal muscle, including insulin responsiveness, which is useful in the research of insulin resistance. The cultured C2C12 cells were thus treated with IL-6 to induce insulin resistance as described previously [12].

After the activation of opioid μ-receptors, tramadol is an effective treatment to handle the pain in clinic with some non-opioid effects [15], while loperamide is a peripheral agonist that does not cross the blood–brain barrier and lacks the side-effects produced by centrally acting opiates [16]. Due to the short half-life of opioid peptides, we used loperamide to evaluate the effect of opioid μ-receptor activation on insulin resistance.

Materials and methods

C2C12 cell culture

The C2C12 cells were obtained from the Culture Collection and Research Center (CCRC 60083) of the Food Industry Institute (Hsin-Chiu City, Taiwan) and cultured as described previously [17]. In brief, cells were plated in 35-mm culture dishes (diameter; 5×104 cells/dish) in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin 10,000 U/ml, streptomycin 10 mg/ml and amphotericin B 25 μg/ml). To induce fusion, confluent cells were exposed to DMEM supplemented with 10% horse serum instead of FBS. Cells were fused into multinucleated myotubes after 7–10 days in culture. Differentiated myotubes were then starved for 5 h in serum-free DMEM before treatment. The medium was changed 24 h prior to experiments.

Glucose uptake

In the previous study [17], maximal 2-[14C]-deoxy-d-glucose (2-DG) uptake was measured in C2C12 cells incubated with 10 μmol/l loperamide. Thus, the same dose of loperamide was used here. After incubation with or without inhibitors (naloxone or naloxonazine) at the indicated concentrations for 30 min, C2C12 cells were treated overnight with loperamide (10 μmol/l) followed by incubation with recombinant human IL-6 (20 ng/ml) for 1 h; this treatment of IL-6 produced a near-maximum effect on insulin resistance [12]. Meanwhile, other groups of cells were incubated with vehicle or IL-6 (20 ng/ml) for 1 h followed by incubation with porcine insulin (1 nmol/l) or vehicle for another 30 min. At the end of the incubation periods, cells were washed with PBS as described previously [17]. The cells were transferred to fresh incubation flasks for incubation with 2-DG (37 kBq/ml) for a further 5 min. Non-specific uptake of 2-DG, assessed after incubation with 20 μmol/l cytochalasin B to block transportation, was subtracted from the total cell-associated radioactivity. Uptake was terminated by adding ice-cold PBS, and cell-associated radioactivity was then determined using a scintillation counter as described previously [17].

Preparation of cell extracts

After incubation overnight with loperamide (10 μmol/l), C2C12 cells were incubated with IL-6 (20 ng/ml) for 1 h followed by incubation with insulin (1 nmol/l) for 3 h. Then, the medium was removed and cells were washed twice with ice-cold PBS. Cell lysates were obtained by scraping the cells in lysate buffer containing 1% Triton X-100, 150 mmol/l NaCl, 10 mmol/l Tris (pH 7.4), 1 mmol/l EDTA, 2 mmol/l NaVO4, 1 mmol/l benzamidine, 0.2 mol/l 4-(2-aminoethyl)benzenesulphonyl fluoride, and 10 μg/ml of antipain, pepstatin and aprotinin. Cell lysates were then spun down, and the cell pellet/debris was discarded. Supernatant protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA).

Immunoprecipitation and immunoblotting

A 1-mg sample of total protein was used for immunoprecipitation with anti-insulin receptor (IR) β-subunit antibody (NeoMarkers, Fremont, CA, USA) or anti-IRS-1 antibody (NeoMarkers) at 4°C overnight. This was followed by the addition of protein A Sepharose beads (Sigma-Aldrich, St Louis, MO, USA) for 1 h. The protein bead antibody complexes were precipitated by brief centrifugation. The pellets were washed three times in ice-cold buffer (0.5% Triton X-100, 100 mmol/l Tris [pH 7.4], 10 mmol/l EDTA, and 2 mmol/l sodium vanadate) and then resuspended in Laemmli sample buffer. The pellets were boiled for 5 min before SDS-PAGE (10% acrylamide gel) with the Bio-Rad Mini-Protein II system (55 V and 130 V for the stacking and separation gels, respectively). Protein was transferred to a polyvinylidene difluoride membrane using the Bio-Rad Trans-Blot system (2 h at 20 V in 25 mmol/l Tris, 192 mmol/l glycine and 20% MeOH). Following transfer, the membrane was probed with anti-IR β-subunit antibody, anti-IRS-1 antibody, or anti-phosphotyrosine antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the instructions of the manufacturers.

For the detection of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase), Akt serine (Ser473) phosphorylation and GLUT-4 content, equal amounts (50 μg) of protein were prepared from cell homogenates and subjected to SDS-PAGE. The protein was transferred to polyvinylidene difluoride membrane as described above and blotted with anti-PI3-kinase p85 subunit antibody (NeoMarkers), anti-phosphoserine (Ser473) Akt antibody (Cell Signaling Technology, Beverly, MA, USA) or anti-GLUT-4 antibody (Genzyme Diagnostics, Cambridge, MA, USA) according to the manufacturer’s instructions. The intensity of the blots incubated with mouse monoclonal antibody to bind the actin (Santa Cruz Biotechnology) was used as control to ensure that the amount of protein loaded into each lane of the gel was constant.

After three 5-min washes in Tris-buffered saline with Tween (20 mmol/l Tris–HCl [pH 7.5], 150 mmol/l NaCl, and 0.05% Tween 20), membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies. The membranes were then washed three times in Tris-buffered saline with Tween and visualised on X-ray film with the enhanced chemiluminescence detection system according to the protocol of the manufacturer (Amersham, Braunschweig, Germany). Densities of the obtained immunoblots were quantified using a laser densitometer.

Statistical analysis

Data were expressed as means±SEM. Statistical differences among groups were determined using two-way repeated-measures ANOVA. The Dunnett range post-hoc comparisons were used and a p value of less than 0.05 was considered statistically significant.

Results

Effect of loperamide on insulin-stimulated glucose uptake

2-DG uptake into C2C12 cells stimulated by 1 nmol/l insulin was 402.5±10.4 pmol·mg protein−1·5 min−1, about 2.7 times the basal uptake (150.8±8.2 pmol·mg protein−1·5 min−1) from samples incubated with DMEM only (Fig. 1). Although basal uptake was slightly reduced by IL-6 (20 ng/ml) to 146.3±9.2 pmol·mg protein−1·5 min−1, the insulin-stimulated 2-DG uptake was markedly impaired by IL-6 (20 ng/ml) to about 50% of that stimulated by insulin alone (Fig. 1). The maximal 2-DG uptake in C2C12 cells by 10 μmol/l of loperamide was 263.8±8.2 pmol·mg protein−1·5 min−1, about 1.8 times the basal uptake (Fig. 1). The insulin-stimulated 2-DG uptake impaired by IL-6 was reversed by loperamide to about 80% of that stimulated by insulin only (Fig. 1). Also, pre-incubation with naloxone or naloxonazine abolished this action of loperamide and achieved a value close to the basal level (Fig. 1).

Fig. 1
figure 1

Effect of loperamide on IL-6-induced inhibition of radioactive 2-deoxyglucose (2-DG) uptake into insulin-stimulated C2C12 cells. One group of C2C12 cells was treated overnight with loperamide (10 μmol/l) followed by incubation with IL-6 (20 ng/ml) for 1 h with or without naloxone (1 μmol/l) or naloxonazine (1 μmol/l) for 30 min. Other groups of cells were incubated with vehicle or IL-6 (20 ng/ml) alone for 1 h followed by incubation with or without insulin for another 30 min. The cells were transferred to fresh incubation flasks with vehicle, loperamide (10 μmol/l), or porcine insulin (1 nmol/l) for 30 min. The cells were incubated with 2-DG (37 kBq/ml) for 5 min to determine glucose uptake. The basal level of glucose uptake was obtained from cells incubated with DMEM only without any treatment. Results are the means±SEM of seven experiments. * p<0.05 and ** p<0.01 vs basal glucose uptake

Full size image

Effect of loperamide on insulin-signalling pathway in insulin-stimulated C2C12 cells

As shown in Fig. 2, insulin increased the protein expression of IR to about 2.6 times the basal level. The degree of IR protein expression in C2C12 cells was slightly decreased by IL-6. Also, insulin-stimulated IR protein expression was reduced by IL-6, showing about 50% of that stimulated by insulin only (Fig. 2). Insulin caused a 2.6-fold increase over basal activity in the degree of IR tyrosine phosphorylation (Fig. 2). However, a combination of IL-6 and insulin decreased IR tyrosine phosphorylation to about 50% of that stimulated by insulin only (Fig. 2). The stimulatory effect of loperamide on IR tyrosine phosphorylation was about 1.6 times the basal level. Incubation of loperamide with IL-6-treated C2C12 cells reversed the degree of IR tyrosine phosphorylation stimulated by insulin to about 2.3 times the basal level (Fig. 2).

Fig. 2
figure 2

Effect of loperamide on IL-6-induced inhibition of insulin signals in C2C12 cells. After incubation with or without naloxonazine (1 μmol/l) for 30 min, C2C12 cells were treated overnight with loperamide (10 μmol/l). Cells were then incubated with IL-6 (20 ng/ml) for 1 h followed by incubation with insulin (1 nmol/l) for 3 h. Other groups of cells were treated with insulin (1 nmol/l) for 3 h or IL-6 (20 ng/ml) for 1 h, followed by incubation with or without insulin for 3 h, or with loperamide (10 μmol/l) alone for 24 h. The representative immunoblots of protein expression and insulin-stimulated phosphorylation in cultured C2C12 cells were analysed by western blot analysis. Similar results were also obtained in the other four experiments. Tyr, tyrosine

Full size image

The protein expression of IRS-1, increased by insulin, was minimised by IL-6 to about 60% of the basal level, and this inhibition was eliminated by loperamide (Fig. 2). In fact, loperamide alone increased the IRS-1 protein expression to about 2.3 times the basal level (Fig. 2). The degree of IRS-1 tyrosine phosphorylation was stimulated by insulin to 2.4 times the basal level and this was reduced by IL-6 to 60% of that stimulated by insulin only (Fig. 2). This effect of IL-6 was reversed by loperamide. Otherwise, loperamide alone elevated the IRS-1 tyrosine phosphorylation to 1.6 times the basal level (Fig. 2).

The expression of the p85 regulatory subunit of PI3-kinase in insulin-stimulated C2C12 cells was increased to 2.5 times the basal level and this was reduced by IL-6 to 50% of that stimulated by insulin only. This inhibition of IL-6 was also markedly reversed by loperamide (Fig. 2). However, loperamide alone did not modify the protein level of the p85 regulatory subunit in C2C12 cells.

An increase of Akt serine (Ser473) phosphorylation by insulin was also observed, showing about four times the basal level (Fig. 2). This phosphorylation, increased by insulin, was also impaired by IL-6 to 50% of that stimulated by insulin only, and this effect of IL-6 was reversed by loperamide (Fig. 2). Treatment with IL-6 alone lowered the phosphorylation to 80% of the basal level. But loperamide alone elevated the phosphorylation to about 1.8 times the basal level (Fig. 2). Moreover, GLUT-4 protein expression was also increased in insulin-treated C2C12 cells, which was about three times the basal level (Fig. 2). IL-6 exerted no effect on the basal level of GLUT-4 but it decreased the insulin-stimulated protein of GLUT-4 to 70% of that stimulated by insulin only, and this effect was markedly reversed by loperamide (Fig. 2). In addition, loperamide alone increased the protein of GLUT-4 to twice the basal level (Fig. 2). Changes in the above immunoblots are quantified and summarised in Table 1.

Table 1 Quantification of the changes in protein expression and insulin-stimulated phosphorylation in C2C12 cells
Full size table

Discussion

Cytokines are implicated in the development of insulin resistance [7, 10, 11]. The negative effect of IL-6 on insulin action was mediated by inhibition of the insulin signalling pathway [14, 18]. Following the previous method [14], 60 min of exposure of IL-6 was used to induce inhibition in this study. Similar to the change in insulin-resistant skeletal muscle [15], insulin stimulation of glucose uptake was impaired by treatment of C2C12 cells with IL-6 for 1 h. This is consistent with the results in hepatocytes and HepG2 cell lines [14]. IL-6 produced in skeletal muscle during contraction is introduced to mediate the training-induced increase of insulin sensitivity in humans [19]. In fact, strenuous exercise imposes an oxidative stress due to the generation of oxygen-free radicals in skeletal muscle [20]. Both reactive oxygen species [21] and lipopolysaccharide [22] can increase IL-6 in skeletal muscle. Exercise alters reactive oxygen species activities in a variety of different ways [23, 24]. Such variable responses to exercise have also been observed in different species [25, 26], and we observed that IL-6 inhibits insulin action to induce insulin resistance in C2C12 cells. These results reinforce the notion that this cytokine is a mediator of insulin resistance.

Loperamide reverses the action of IL-6 to restore the insulin-mediated glucose utilisation identified by the glucose uptake. This action of loperamide was inhibited by naloxone or naloxonazine at doses sufficient to block opioid μ-receptors, indicating that the recovery of insulin action by loperamide is induced by activation of opioid μ-receptors. Thus, we used the response to loperamide as an indicator of opioid μ-receptor activation to investigate the possible changes in insulin signalling.

In the present study, IL-6 impaired the insulin signalling pathway from IR; both the protein expression and the degree of autophosphorylation were affected. In fact, certain insulin signals became defective in insulin resistance [27]. In insulin-stimulated glucose uptake, IRS-1 and PI3-kinase are believed to be the critical signals [28], but they are also reduced by IL-6. Usually, the IRS-1 tyrosine phosphorylation stimulated by insulin increases IRS-1 association with p85, the regulatory subunit of PI3-kinase [28]. We observed that the p85 level is markedly reduced in IL-6-treated C2C12 cells. The PI3-kinase activation is required for insulin-mediated glucose uptake [29], because impaired PI3-kinase activation is linked with decreased GLUT-4 translocation [30] and insulin resistance [31]. Moreover, the downstream signal of PI3-kinase is introduced as the serine/threonine kinase protein B (also known as Akt) [32]. IL-6 inhibits the insulin-stimulated Akt serine phosphorylation, consistent with an impairment of Akt serine phosphorylation in animals exhibiting insulin resistance [33]. Also, similar to the decrease of GLUT-4 in insulin resistance [34], a decrease of insulin-stimulated GLUT-4 protein is observed in the membrane fraction of IL-6-treated C2C12 cells. The reduction in insulin signalling by IL-6 is obtained and this is consistent with the inhibition of insulin action identified by the glucose uptake.

The reduction in insulin signalling by IL-6 was reversed by loperamide; all defective insulin signals, as above, were ameliorated by opioid μ-receptor activation. The recovery of insulin action by opioid μ-receptor activation seems to be related to this normalisation of insulin signals. However, as opposed to IRS-1, IRS-2 is expressed to a lesser extent in skeletal muscle [35]. Although IL-6 has been shown to play a minor role in the defects of IRS-2-associated PI3-kinase activity [36], an effect of the activation of opioid μ-receptors on the IRS-2-related signals cannot be excluded; more investigations are required. Taken together, these observations show that opioid μ-receptor activation reverses the action of insulin by increasing the genes involved in insulin signalling pathways, specifically the IRS-1-associated PI3-kinase step.

In fact, β-endorphin can reverse insulin action by an activation of opioid μ-receptors to enhance the PI3-kinase/Akt activity [8]. Thus, activation of opioid μ-receptor conferred a beneficial effect on insulin resistance. Recently, the therapy of type 2 diabetes has seen a shift to insulin sensitiser and thiazolidinediones [37], because insulin sensitiser has the ability to improve insulin sensitivity [38]. However, an insulin-sensitising effect of opioid μ-receptor activation cannot be ruled out. The development of agents to activate peripheral opioid μ-receptors without opiate-like effects on the central nervous system will be useful for patients with insulin resistance.

Protein kinase C (PKC) activation by a phospholipase-C- (PLC) dependent mechanism is the main signal linked to opioid μ-receptors and has been shown to mediate morphine-induced supraspinal antinociception [39]. The PLC–PKC pathway is also involved in the mechanism of opioid tolerance [40] and the insulin signals linked to glucose transportation [17]. Thus, the PLC–PKC pathway seems to be responsible for the cross-talk of opioid μ-receptor activation with insulin signals. In fact, atypical PKCs are essential for insulin stimulation of glucose transport in muscle tissue [41]. Although atypical PKCs are located downstream of IRS-1 and PI3-kinase in insulin signalling, IRS-1 is also a novel substrate for atypical PKCs [42]. Moreover, suppressors of cytokine signalling (SOCS) proteins have been implicated in the insulin signalling network [43]. SOCS-3 can inhibit insulin signalling [44], and the negative effect of IL-6 on insulin signalling is related to the higher gene expression of SOCS-3 [45]. The role of SOCS-3 or atypical PKCs in the effect of opioid μ-receptor activation requires due attention. Nevertheless, our data clarify the important role of opioid μ-receptor activation in the improvement of insulin resistance induced by cytokines.

In conclusion, activation of opioid μ-receptors by loperamide amends the impairment of insulin-stimulated glucose uptake in IL-6-treated C2C12 cells. This improvement is associated with a marked recovery of the reduced insulin signals and/or impaired insulin action by IL-6. These data strengthen the basis for recommending peripheral opioid μ-receptor activation as a new target for improving insulin action.