Introduction

Insulin resistance develops when tissues in the body (skeletal muscle, adipose/fat, and liver) become less sensitive and ultimately resistant to insulin, the hormone that is produced by the β-cells in the pancreas [1, 2]. Furthermore, insulin resistance is a major defect underlying the development of type II diabetes and is a central factor in the development of the metabolic syndrome, a group of abnormalities including obesity and hypertension [3, 4]. Among all the tissues in the body, the skeletal muscle tissue is majorly responsible, accounting for 75–80 %, for insulin-stimulated glucose uptake in the postprandial state; it also plays a pivotal role in maintaining glucose homeostasis [5, 6]. In skeletal muscle tissue, insulin stimulates glucose uptake primarily by increasing the translocation of the glucose transporter-4 (GLUT4) from the internal membrane to the plasma membrane [7, 8]. The signaling mechanism by which insulin stimulates muscle glucose uptake is well known and involves binding of insulin to its receptor, phosphorylation of downstream insulin receptor substrates (IRS), and activation of phosphatidylinositol-3 kinase (PI3-K) and protein kinase B (Akt), which promotes GLUT4 translocation from a cellular pool in the plasma membrane [5, 9, 10]. AMP-activated protein kinase (AMPK) complex, a αβγ heterotrimer that functions as a cellular energy sensor, has been positively correlated with increased muscle glucose uptake, and in recent years, it has emerged as an attractive pharmacological agent for the treatment of insulin resistance and type II diabetes [5, 11, 12]. In skeletal muscles, AMPK is activated by exercise/contraction and numerous compounds including thiazolidinediones [13], metformin [14], and resveratrol [5], resulting in the stimulation of glucose uptake. The study of new compounds that activate AMPK and stimulate skeletal muscle glucose uptake is important, because the knowledge obtained from such studies could be used toward the development of treatments for insulin resistance and type II diabetes [6].

Hypertension is associated with insulin resistance and glucose intolerance. Increased skeletal muscle blood flow and the resulting improvements in insulin delivery are important mechanisms by which attenuation of the renin–angiotensin system (RAS) improves glucose uptake [15]. One potential mechanism involves the inhibition of angiotensin I-converting enzyme (ACE), which increases nitric oxide (NO) generation and enhances glucose transportation activity in skeletal muscles [15, 16]. In our previous studies, we purified and identified an antihypertensive peptide derived from S. clava flesh tissue and successfully demonstrated its ACE inhibitory effect and NO-mediated vasorelaxation [17, 18]. In the present study, we examined the effects of the antihypertensive peptide on glucose uptake and GLUT4 translocation and attempted to elucidate the mechanism of action underlying the antihypertensive peptide’s effects in L6 skeletal muscle cells.

Materials and methods

Materials

The rat myoblast cell line L6 was purchased from the Korean Cell Line Bank (KCLB; Seoul, KOREA). DMEM (Dulbecco’s modified Eagle’s medium), wortmannin, and compound C were purchased from Sigma (St. Louis, MO, USA). Antibodies against AMP-activated protein kinase (AMPK), phospho-AMPK (Thr172), and glucose transporter-4 (GLUT4) were from Cell Signaling Technology (Bedford, Massachusetts, USA). Second IgG HRP-linked antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The other chemicals and reagents used were of analytical grade.

Purification and identification of the purified peptide and peptide synthesis

To extract the purified peptide from S. clava flesh tissue, a hydrolysis was performed using Protamex, a commercial food grade enzyme, under optimal conditions [17]. The Protamex hydrolysate was fractionated through ultrafiltration membranes with molecular weight (MW) cutoff of 5 kDa. The below 5 kDa fraction was again filtered and applied to a column saturated in Sephadex G-25 resin, which was previously equilibrated with distilled water. Further fractions were collected with a fraction collector. The fraction exhibiting the highest antihypertensive effect was further purified using reverse-phase HPLC on an octadecyl silica (ODS) column. Finally, the fraction with the greatest antihypertensive effect was collected; this was followed by identification of the amino acid sequence. Amino acid sequence of the purified peptide was identified as Ala-His-Ile-Ile-Ile (AHIII), with a molecular weight 565.3 Da.

Synthesis of antihypertensive peptide

In order to validate the glucose uptake of the antihypertensive peptide, a synthetic peptide with the same sequence was synthesized and tested. The antihypertensive peptide was chemically synthesized in the peptide synthesizer facility PepTron Inc. (Daejeon, Korea). The peptide was synthesized using the Fmoc solid-phase method with a peptide synthesizer (PeptrEX-R48, Peptron Inc., Daejeon, Korea). The synthetic peptide was purified using HPLC (SHIMADZU, Kyoto, Japan) on a Capcell Pak C18 column (Shiseido, Tokyo, Japan). The column was developed at a flow rate of 1.0 mL/min by a linear gradient of acetonitrile (0–20 %, 2 min; 20–50 %, 10 min; 50–80 %, 2 min) containing 0.1 % trifluoroacetic acid. The identity of peptide was confirmed by mass analysis using LC–MS (Agilent Technologies, CA, USA), and the purity of synthetic peptide was confirmed over 95 %.

Cell culture

Rat myoblast L6 cells were maintained in high-glucose DMEM supplemented with 10 % heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). Cultures were maintained at 37 °C in 5 % CO2 incubator. For differentiation, the cells were seeded in appropriate culture plates, and after sub-confluence (about 80 %), the medium was changed to DMEM containing 2 % horse serum for 7 days, with medium changes every day. All experiments were performed in differentiated L6 myotubes after 7 days.

MTT assay

The cytotoxicity of antihypertensive peptide against the L6 cells was determined by a colorimetric MTT assay. Cells were seeded in a 24-well plate. After 24 h, the cells were treated with various concentrations (25, 50, 100, and 200 μM) of the antihypertensive peptide. The cells were then incubated for an additional 24 h at 37 °C. MTT stock solution (100 μL; 2 mg/mL in PBS) was then added to each well. After incubating for 4 h, the plate was centrifuged at 2000 rpm for 10 min and the supernatant was aspirated. The formazan crystals in each well were dissolved in DMSO. The amount of purple formazan was determined by measuring the absorbance at 540 nm.

Glucose uptake assay

L6 cells were seeded in a 24-well plate. After differentiation, the cells were starved in serum-free low-glucose DMEM for 12 h and then washed with PBS and incubated with fresh serum-free low-glucose DMEM. After that, the cells were treated with insulin (100 nM) for 1 h, or the indicated concentrations (to determine the dose response of L6 myotubes to antihypertensive peptide) of antihypertensive peptide. Glucose uptake was measured by glucose concentration in the medium solution using glucose oxidase assay kit (Asan Pharmaceutical corp., Korea).

In some experiments, 100 nM of Wortmannin (PI3 kinase inhibitor) and 10 μM of compound C (AMPK inhibitor) were added 30 min before the antihypertensive peptide treatment.

Western blot analysis

L6 myotubes were grown in 100-mm dishes and were starved in serum-free low-glucose DMEM for 12 h prior to treatment with the indicated agents. Following treatment, the media were aspirated and the cells were washed twice in ice-cold PBS. The cell lysates were prepared with lysis buffer (20 mM Tris, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1 % NP-40, 25 μg/mL aprotinin, 25 μg/mL leupeptin, and 1 mM PMSF) for 60 min and then centrifuged at 12,000 rpm for 15 min at 4 °C. The protein concentrations were determined by using BCA™ protein assay kit. The lysate containing 30 μg of protein was subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gel, and the gel was transferred onto a nitrocellulose membrane. The membranes were blocked in 5 % nonfat dry milk in TBST (25 mM Tris–HCl, 137 mM NaCl, 2.65 mM KCl, 0.05 % Tween 20, pH 7.4) for 2 h. The primary antibodies were used at a 1:1000 dilution. Membranes incubated with the primary antibodies at 4 °C for overnight. And then the membranes were washed with TBST and then incubated with the secondary antibodies used at 1:3000 dilutions. Signals were developed using an ECL Western blotting detection kit and exposed to X-ray films.

Plasma membrane fractionation and immunoblot analysis

L6 myotubes were treated with the indicated agents and harvested. The cell lysates were prepared with lysis buffer [250 mM sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), and protease inhibitors (1 mM PMSF, 25 μg/mL aprotinin, and 25 μg/mL leupeptin)] and kept on ice for 10 min. The cell lysate was ultracentrifuged at 22,000 rpm for 1 h at 4 °C. The pallet was re-suspended in a lysis buffer and kept on ice for 10 min and then centrifuged at 8000 rpm for 5 min at 4 °C to obtain plasma membrane fraction from the middle layer of the supernatant. Immunoblot analyses of GLUT4 described in the method to 2.7.

Statistical analysis

All data were represented as the mean ± SD. Statistical comparisons of the mean values were performed by analysis of variance (ANOVA), followed by Duncan’s multiple-range test using SPSS (11.5) software. Statistical significance was considered at p < 0.05.

Results

The antihypertensive peptide, AHIII, does not affect the viability of L6 skeletal muscle cells

Prior to evaluating the glucose uptake of the antihypertensive peptide, we first examined its cytotoxic effect on the viability of L6 skeletal muscle cells. Cytotoxicity of different concentrations of the peptide AHIII (25, 50, 100, and 200 μM) was evaluated using the MTT assay. Compared to the control, AHIII did not have significant cytotoxic effects on L6 skeletal muscle cells (Fig. 1). Based on these findings, the above-mentioned concentrations were used for subsequent experiments.

Fig. 1
figure 1

Cytotoxicity of the antihypertensive peptide was determined using the MTT assay. Each value is expressed as mean ± standard deviation in triplicate experiments

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The antihypertensive peptide stimulates glucose uptake in dose-dependent manner

Differentiated L6 skeletal muscle cells were used to determine the role of the peptide in the glucose metabolism. It was found that AHIII dose-dependently stimulated glucose uptake as shown in Fig. 2, and the effect at a 200 μM concentration was comparable to that of insulin; the antihypertensive peptide may have metabolic effects in skeletal muscle cells.

Fig. 2
figure 2

Antihypertensive peptide dose-dependently stimulates glucose uptake in L6 skeletal muscle cells. Cells were starved in serum-free media for 6 h and incubated for 1 h with increasing in antihypertensive peptide and insulin. The values are expressed as the mean ± standard deviation of triplicate experiments. *p < 0.05 indicate significant differences compared with the untreated group

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The antihypertensive peptide activates AMPK pathway in L6 skeletal muscle cells

To investigate the pathway responsible for the effect of the antihypertensive peptide on glucose uptake in L6 cells, the cells were pretreated with wortmannin, an inhibitor of phosphatidylinositol (PI)-3 kinase and compound C, a selective AMPK inhibitor. As shown in Fig. 3, compound C significantly inhibited the glucose uptake stimulated by peptide AHIII (200 μM). Further, the effect of AHIII on AMPK activation was investigated. We found that AHIII induced increased AMPK phosphorylation in L6 skeletal muscle cells (Fig. 4). However, the increase in AMPK phosphorylation was inhibited by a 30-min pretreatment of the cells with compound C (Fig. 4). Taken together, the findings strongly indicate that the antihypertensive peptide plays a metabolic role in skeletal muscle cells via the AMPK pathway.

Fig. 3
figure 3

Antihypertensive peptide-induced increase in glucose uptake was reduced by wortmannin and compound C. After 6-h starvation, L6 skeletal muscle cells were pretreated with or without 100 nM wortmannin (phosphatidylinositol (PI)-3 kinase inhibitor) and 10 μM compound C (AMPK inhibitor) for 30 min and then treated with 200 μM of antihypertensive peptide for 1 h. The values are expressed as the mean ± standard deviation of triplicate experiments. *P < 0.05 versus untreated group or between two groups as indicated

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Fig. 4
figure 4

Effect of the antihypertensive peptide on AMPK signaling pathway. Cells were pretreated with or without 10 μM compound C for 30 min and then treated with the indicated concentrations of the antihypertensive peptide and insulin for 1 h and 10 min, respectively. The cell lysates were analyzed via Western blotting using anti-phospho-AMPK (Thr172) and anti-AMPK. The values are expressed as the mean ± standard deviation of triplicate experiments. *P < 0.05 versus untreated group or between two groups as indicated

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The antihypertensive peptide stimulates glucose uptake by increasing GLUT4 translocation through the AMPK pathway

We examined the effect of the antihypertensive peptide on the AMPK signaling pathway responsible for the translocation of GLUT4 to the plasma membrane and increased glucose uptake. After L6 skeletal muscle cells were treated with peptide AHIII for 4 h, GLUT4 translocation was determined. As seen in Fig. 5, AHIII treatment markedly increased GLUT4 translocation to the plasma membrane of L6 skeletal muscle cells. However, the increased GLUT4 translocation to the plasma membrane of AHIII-treated L6 skeletal muscle cells was almost completely abolished by compound C pretreatment. These results suggest that the antihypertensive peptide stimulates GLUT4 translocation to the plasma membrane possibly via activation of the AMPK pathway.

Fig. 5
figure 5

Effect of the antihypertensive peptide on GLUT4 translocation to the plasma membrane. Cells were pretreated with or without 10 μM compound C for 30 min and then treated with the indicated concentrations of the antihypertensive peptide and insulin for 1 h and 10 min, respectively. The cell lysates were analyzed via Western blotting using anti-GLUT4. The values are expressed as the mean ± standard deviation of triplicate experiments. *P < 0.05 versus untreated group or between two groups as indicated

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Discussion

Type II diabetes poses a major threat to human health worldwide and is the most common type of diabetes [19]. The available drugs for type II diabetes have a number of limitations, including adverse effects and high rates of secondary failure. In recent years, biological resources have become the focus of a considerable amount of attention from researchers searching for alternative therapies for diabetes [20, 21] because they are typically considered less toxic and to have fewer side effects than synthetic drugs do. Hence, the identification and characterization of bioactive peptides from biological sources have become increasingly important and are of great interest to researchers [22].

Generally, skeletal muscles play an important role in the regulation of energy balance and glucose metabolism [23]. Hence, L6 skeletal muscle cells were used to evaluate the antidiabetic properties of the antihypertensive peptide derived from S. clava flesh tissue. To our knowledge, this is the first report of an antihypertensive peptide that significantly stimulates glucose uptake. It is important to note that this study is particularly relevant owing to the effects observed in skeletal muscle cells.

As mentioned previously, glucose uptake in skeletal muscle tissue can be mediated by at least two major mechanisms: (1) through insulin-dependent activation of PI3-K/Akt and (2) the activation of AMPK by muscle contraction due to exercise in order to maintain the energy balance [24, 25]. Our findings in L6 skeletal muscle cells show that a significant increase in glucose uptake occurs owing to the antihypertensive peptide via the PI3-K/Akt pathway in the absence of insulin. In the presence of wortmannin, a selective PI3-K/Akt inhibitor, the antihypertensive peptide did not affect the PI3-K/Akt pathway. In contrast, compound C significantly reduced glucose uptake in L6 skeletal muscle cells.

AMPK plays a key role in energy homeostasis in ATP-depleting metabolic states such as ischemia, hypoxia, heart shock, oxidative stress, and especially exercise [26, 27]. Once activated under such conditions, it accelerates ATP-generating catabolic pathways including glucose uptake and fatty acid oxidation through direct regulation of key metabolic enzymes [28]. In recently published studies, it has been reported that AMPK serves as a key metabolic sensor through cellular regulation of insulin-independent glucose uptake and glycogen metabolism [29]. Thus, AMPK is emerging as a potential target for the treatment of diabetes, especially because it could play a principal role in the exercise-induced adaptation of skeletal muscles, type II diabetes, obesity, and the metabolic syndrome [23, 30]. The present study showed a significant increase in AMPK phosphorylation by the antihypertensive peptide. Further, the antihypertensive peptide-mediated activation of AMPK is abolished by pretreatment of compound C, a highly selective AMPK inhibitor. Additionally, the peptide-induced increase in GLUT4 translocation to the plasma membrane suggests its involvement in AMPK regulation of glucose metabolism in L6 skeletal muscle cells.

In summary, these results demonstrate that the antihypertensive peptide improves glucose uptake via activation of the AMPK pathway in skeletal muscles. In particular, skeletal muscles play a major role in the regulation of energy balance [29] and are the primary tissues involved in glucose uptake and disposal.

In conclusion, the antihypertensive peptide increases glucose uptake via activation of the AMPK pathway and may potentially emerge as a novel target for the treatment of type II diabetes.