2015 年 21 巻 5 号 p. 727-732
Soy β-conglycinin (βCG) consumption has been shown to improve insulin resistance and suppress diet-induced obesity. These physiological effects seem to be mediated by the normalization of adiponectin and insulin sensitivity. Here, we show that artificial enzyme-hydrolyzed βCG peptides promote uptake of 2-deoxyglucose, a glucose analogue, accompanied by translocation of glucose transporter 4 in skeletal L6 myotubes. Inhibition of AMP-activated protein kinase (AMPK) attenuated βCG peptides-induced glucose uptake activity, while inhibition of both insulin and nitrogen monoxide signaling did not affect the glucose uptake activity. Taken together, these results indicate that βCG peptides directly induced glucose uptake through the AMPK signaling pathway in muscle cells and might function to prevent insulin resistance.
Glucose transporters (GLUTs) play an important role in the regulation of blood glucose levels. GLUT4 is specifically expressed in skeletal muscle and adipose tissue, and is mainly localized in intracellular storage vesicles. GLUT4 storage vesicles translocate to the plasma membrane in response to various stimuli and uptake glucose to reduce postprandial hyperglycemia (Belman, et al., 2014). In skeletal muscle, the translocation is regulated by insulin and AMP-activated protein kinase (AMPK) signaling pathways (Sheena, et al., 2011). Activation of AMPK is induced by the following three mechanisms: allosteric activation, phosphorylation of α subunit Thr172, and inhibition of Thr172 dephosphorylation (Kahn, et al., 2005). The degree of AMPK activation depends on the intensity of exercise and is thought to be induced by changes in AMP/ATP and creatine/phosphocreatine ratios. Nitric oxide (NO) is also thought to regulate AMPK activation during exercise (Merry, et al., 2009). It has been reported that AMPK is activated by the anti-diabetic drugs metformin (Zhou, et al., 2001) and thiazolidinediones (Fryer, et al., 2002). Thiazolidinediones increase the AMP/ATP ratio in L6 myotubes to stimulate AMPK activity, whereas metformin induces phosphorylation of AMPK without changing ATP levels in the cells (Hawley, et al., 2002). These reports suggest that AMPK is a major target molecule for anti-diabetic therapy.
Many reports have demonstrated that soy protein possesses various beneficial effects. For example, soy protein produces a hypocholesterolemic effect and may help to prevent heart disease (Hamilton, et al., 1976, U.S. Food Drug Administration). Consuming a diet rich in soy protein has been reported to increase insulin sensitivity and plasma insulin levels and decrease fasting blood glucose levels in diabetic model rodents (Cederroth, et al., 2008; Lu, et al., 2008). In addition, soy protein hydrolysates derived from pepsin digestion have been shown to lower blood pressure and suppress angiotensin-converting enzyme (ACE) activity in rats with Nω-nitro-l-arginine methyl ester hydrochloride-induced hypertension (Yang, et al., 2008). This result suggests that soy protein peptides contribute to the beneficial effects of soy protein.
β-Conglycinin (βCG) is one of the main storage proteins in soy and accounts for 27 – 40% of total soy protein. It has been reported that consumption of βCG has an anti-obesity effect through the reduction of plasma triglycerides (Tachibana, et al., 2010; Kohno, et al., 2006). In addition, our group recently reported that consumption of βCG promotes GLUT4 translocation through the AMPK signaling pathway in a rat model of diabetes (Tachibana, et al., 2014). Adiponectin is known as an AMPK activator (Kadowaki and Yamauchi, 2005), and βCG increases adiponectin levels in serum and its expression level in visceral fat (Tachibana, et al., 2014). However, AMPK activation is not observed in the livers of mice fed with βCG. These reports hypothesized that βCG might directly influence glucose metabolism in skeletal muscle. Dietary proteins are hydrolyzed into peptides and free amino acids in the alimentary canal. To clarify the mechanism of βCG on glucose metabolism in skeletal muscle, we prepared βCG peptides by digesting βCG with an artificial mixture of gastrointestinal enzymes and investigated the effect of these peptides on glucose uptake in rat L6 myotubes.
Chemicals and antibodies Pepsin, trypsin, chymotrypsin, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 2-deoxyglucose (2DG), glucose-6-phosphate dehydrogenase (G6PDH), 7-hydroxy-3H-phenoxazin-3-one 10-oxide (resazurin), Compound C, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), and LY294002 were purchased from Sigma (St. Louis, MO, USA). Diaphorase and β-nicotinamide adenine dinucleotide phosphate (NADP)+ were from Oriental Yeast (Tokyo, Japan). NG-monomethyl-L-arginine (L-NMMA), monoacetate salt was from Calbiochem (La Jolla, CA, USA).
For western blotting, anti-GLUT4 goat IgG, anti-goat IgG, and anti-rabbit IgG antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); anti-AMPKα rabbit IgG, and anti-phospho-AMPK-α (Thr 172) rabbit IgG were from Cell signaling Technology Co. (Danvers, MA, USA). All other reagents used were of the highest grade available commercially.
Preparation of βCG peptides Peptides were prepared from βCG (Lipoff, Fuji oil Co., Osaka, Japan) as follows: βCG was dissolved in water at 2.5% and hydrolyzed by pepsin (1:100) for 2 h under acidic conditions of pH 2 at 37°C. After that, βCG solution was degraded for 2 h using trypsin (1.7% per substrate) and chymotrypsin (0.03% per substrate) under neutral conditions of pH 8 at 37°C. After enzymatic hydrolysis, βCG solution was boiled at 90°C for 5 min and centrifuged at 10,000 × g for 10 min. The supernatant (βCG peptides fraction) was recovered, snap-frozen in liquid nitrogen, and lyophilized. Freeze-dried sample was subsequently referred to as a βCG peptides fraction and stored at −20°C until use.
Measurement of molecular size of βCG peptides The molecular weights of βCG peptides were calculated using a modified method described in a previous report (Mabuchi and Nakahashi, 1981). Two gel filtration columns with different fractionation ranges, a TSK G3000SWXL gel column (7.8×300 mm, Tosoh Co., Tokyo, Japan) followed by a G2000SWXL column (7.8×300 mm, Tosoh), were connected. Elution was performed at a flow rate of 0.4 mL/min with elution buffer [50 mM phosphate butter, pH 7, 1.17% (w/v) NaCl, 1% (w/v) sodium dodecyl sulfate] at room temperature. The detection of peptides was measured at 220 nm.
Cell culture and treatment with βCG peptides A rat myoblast cell line (L6) was purchased from Sumitomo Dainippon Pharma Co., Ltd (Tokyo, Japan) and maintained in DMEM supplemented with 10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin at 37°C in a 5% CO2 atmosphere. When L6 myoblasts reached confluence, the medium was replaced every 2 days with differentiation medium consisting of MEM supplemented with 2% FBS. Fully differentiated myotubes (8 days after differentiation) were used for subsequent experiments. After the cells were serum-starved for 16 h in MEM containing 0.2% BSA, the cells were incubated with indicated concentrations of βCG peptides for 4 h and then immediately washed twice with ice-cold Krebs-Ringer phosphate-HEPES buffer (KRH; 50 mM HEPES, pH 7.4, 137 mM NaCl, 4.8 mM KCl, 1.85 mM CaCl2, and 1.3 mM MgSO4). Cells treated with 100 nM insulin for 15 min were used as a positive control.
Measurement of glucose uptake Serum-starved L6 myotubes in a 96-well plate were treated with βCG peptides and/or other compounds in 0.2% (w/v) BSA/DMEM. The final concentration and the time of incubation of each compound are indicated in the figures. For the inhibition of signal pathways, L6 myotubes were pre-incubated with 20 µM Compound C (an AMPK inhibitor), 10 µM LY294002 (a PI3K inhibitor), or 0.1 mM L-NMMA (a NOS inhibitor) for 16 h, 30 min, or 5 min, respectively. As a positive control, cells were treated with 100 nM insulin for 4 h. DMSO was used as a vehicle control (final concentration was 0.1%). Cells were further incubated with 1 mM 2DG for 20 min in KRH containing 0.1% (w/v) BSA. The amount of 2DG incorporated into the cells was enzymatically determined as described previously (Yamamoto, et al., 2006; 2010). Briefly, cells were washed twice with 0.1% (w/v) BSA/KRH buffer, lysed with 0.1 N sodium hydroxide, warmed at 60°C for 10 min, and dried at 85°C for 50 min. The dried cell lysate was solubilized with 0.1 N hydrochloric acid and 200 mM triethanolamine (TEA) (pH 8.1), and gently stirred using a microplate shaker. The lysate was mixed with an assay cocktail [50 mM TEA, pH 8.1, 50 mM KCl, 0.02% (w/v) BSA, 0.1 mM NADP+, 2 units diaphorase, 150 units G6PDH, and 2 µM resazurin] on another 96-well plate and incubated at 37°C for 50 min. The fluorescence of resorufin was measured at 570 nm with excitation at 530 nm using a Wallac 1420 ARVOsx (Perkin-Elmer, Boston, MA, USA). The 2DG concentration in each well was calculated based on a standard curve generated with a 2DG-6-phosphate solution.
Western blot analysis Plasma membrane fractions and cell lysates from L6 cells were prepared as previously described (Nishiumi and Ashida, 2007). To prepare the plasma membrane fraction, cells were harvested with buffer A [50 mM Tris, pH 8.0, 0.5 mM dithiotheritol (DTT), protease and phosphatase inhibitors] containing 0.1% (v/v) Nonidet P-40 (NP-40), and centrifuged at 1,000 × g for 10 min at 4°C. The precipitate was suspended in buffer A, placed on ice for 10 min, and centrifuged at 1,000 × g for 10 min at 4°C. The precipitate was suspended in buffer A containing 1.0% NP-40, placed on ice for 1 h, and centrifuged at 16,000 × g for 20 min at 4°C. The supernatant was collected and stored as the plasma membrane fraction. To prepare cell lysate, cells were harvested with a lysis buffer [10 mM Tris, pH 8.0, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% sodium dexycholate, 0.1% sodium dodecyl sulfate (SDS), and 0.5 mM DTT] containing protease and phosphatase inhibitors, placed on ice for 1 h, and centrifuged at 16,000 × g for 20 min at 4°C. The supernatant was collected and stored as the cell lysate. The plasma membrane fraction was used for the detection of GLUT4 translocation and the expression of IRβ. Cell lysate was used for the detection of the expression and phosphorylation levels of proteins related to GLUT4 translocation. Primary and secondary antibodies were diluted 1:10,000 and 1:20,000, respectively in Can Get Signal (Toyobo Co., Ltd., Osaka, Japan). The protein bands were visualized using ImmunoStar LD (Wako Pure Chemical Industries, Osaka, Japan) and detected with a light-Capture II (ATTO Corp., Tokyo, Japan). Densities of specific bands were determined using ImageJ image analysis software provided by the National Institutes of Health (NIH, Bethesda, MD, USA).
Statistical analysis Statistical analyses were performed with factorial ANOVA with the Tukey-Kramer multiple-comparison test as a post hoc test. Data are expressed as means ± SE. The level of significance was defined as p < 0.05.
Effects of βCG peptides on glucose uptake activity and GLUT4 translocation in L6 myotubes. βCG polypeptides are 7S trimers with molecular masses of (150 – 175)×103 and are comprised of various combinations of three non-identical but homologous polypeptide subunits: the α′, α, and β subunits with Mr = 76,000, 72,000, and 53,000, respectively (Yaklich, 2001). The molecular weights of βCG peptides used in this study ranged from 200 to 150,000, and the main peak of the peptides was about 5000 (Figure 1). Next, we investigated whether βCG peptides promote glucose uptake activity and GLUT4 translocation in L6 myotubes (Figures 2 and 3). βCG peptides increased the glucose uptake activity in a dose-dependent manner, and a significant increase was observed at 3 and 10 mg/mL. βCG peptides at 3 mg/mL had almost the same effect as insulin at 100 nM (Figure 2). As shown in Figure 3, βCG peptides also significantly promoted GLUT4 translocation compared with the control cells. βCG peptides used in this study did not show any cytotoxicity under our experimental conditions (data not shown). These results indicate that βCG peptides are able to increase glucose uptake accompanied by GLUT4 translocation to the plasma membrane in L6 myotubes.
Molecular distribution of βCG peptides.
HPLC was performed as described in the Materials and Methods. Molecular weight markers were used as follows: p-aminobenzoic acid (MW, 137; Rt, 63.6), glutathione (MW, 307; Rt 57.7), insulin (MW, 5734; Rt, 43.6), cytochrome-C (MW, 12384; Rt, 41.5), myoglobin (MW, 18000; Rt, 38.2), peroxidase (MW, 43000; Rt, 34.8), albumin (MW, 67000; Rt, 32.3), γ- globulin (MW, 150000; Rt, 29.7), and thyroglobulin (MW, 335000; Rt, 28.3). The approximation formula was y = −4.611ln(x) + 84.588, and the correlation coefficient (R) was 0.9945.
Effects of βCG peptides on glucose uptake in L6 myotubes.
Glucose uptake was measured in L6 myotubes treated with 0.3 – 10 mg/mL βCG peptides for 4 h. The cells were also treated with DMSO and 100 nM insulin as the negative and positive controls, respectively. Values are represented as means ± SE (n=3). Different letters indicate significant differences between the groups (p < 0.05; Tukey HSD test).
Effects of βCG peptides on GLUT4 translocation.
βCG peptides at 1 and 10 mg/mL were treated to L6 myotubes for 4 h. DMSO and 100 nM insulin were also treated to the cells as negative and positive controls, respectively. GLUT4 translocation from storage vesicles (cell lysate) to the plasma membrane was measured by western blot analysis as described in the Materials and Methods. Band density was determined by ImageJ analysis software. Values are represented as means ± SE (n=3). Different letters indicate significant differences between the groups (p < 0.05; Tukey HSD test).
βCG peptides promoted glucose uptake through the AMPK-dependent pathway in L6 myotubes. In skeletal muscle, GLUT4 translocation is regulated by AMPK activation (Sheena, et al., 2011). As shown in Figure 4A, βCG peptides and AICAR, a specific activator of AMPK, strongly increased phosphorylation of AMPK. To confirm whether βCG peptides promote glucose uptake through AMPK activation, Compound C was introduced as an AMPK inhibitor (Figure 4B). Compound C significantly suppressed both βCG peptide- and AICAR-induced glucose uptake in L6 myotubes. These results indicate that βCG peptides promoted glucose uptake through the AMPK-dependent pathway in skeletal muscle cells.
Effects of βCG peptides on activation of AMPK and glucose uptake in L6 myotubes with AMPK inhibitor.
(A) L6 myotubes were treated with βCG peptides at 1 and 10 mg/mL for 4 h. DMSO and 2 mM AICAR were used as controls. AMPK phosphorylation was estimated by western blot analysis as described in the Materials and Methods. Band density was determined by ImageJ analysis software. (B) For measurement of glucose uptake, L6 myotubes were treated with 10 mg/mL βCG peptides with or without Compound C as an inhibitor of AMPK. DMSO and 2 mM AICAR were used as controls. Values are represented as means ± SE (n=3). Different letters indicate significant differences between the groups (p < 0.05; Tukey HSD test).
PI3K and NO were not involved in βCG peptides-induced glucose uptake in L6 myotubes. In skeletal muscle, insulin also promotes GLUT4 translocation through the PI3K-dependent pathway (Sheena, et al., 2011). To clarify whether PI3K is involved in βCG peptide-induced glucose uptake activity, LY294002 was introduced as a PI3K inhibitor (Figure 5). Glucose uptake in L6 myotubes was similar in response to 10 mg/mL βCG peptides and 100 nM insulin, but an additive effect was not observed. LY294002 decreased glucose uptake in cells treated with insulin to the control level, but this specific inhibitor did not affect glucose uptake in βCG peptide-treated cells. From this result, we concluded that βCG peptides promoted glucose uptake through PI3K-independent pathway in L6 myotubes.
Effects of PI3K inhibitor on βCG peptide-promoted glucose uptake in L6 myotubes.
L6 myotubes were treated with βCG peptides at 10 mg/mL for 4 h with or without LY294002 as a PI3K inhibitor. DMSO and 100 nM insulin were used as controls. Values are represented as means ± SE (n=3). Different letters indicate significant differences between the groups (p < 0.05; Tukey HSD test).
AMPK activation is regulated by various biological factors, and NO is one of the activators for AMPK in skeletal muscle (Merry, et al., 2009). To clarify whether NO is involved in βCG peptide-induced glucose uptake activity, L-NMMA was introduced as an eNOS inhibitor (Figure 6). L-NMMA did not affect βCG peptides-induced glucose uptake activity, suggesting that NO was not involved in βCG peptides-induced glucose uptake in L6 myotubes.
Effects of nitric oxide synthetase inhibitor on βCG peptide-promoted glucose uptake in L6 myotubes.
L6 myotubes were treated with βCG peptides at 10 mg/mL for 4 h with or without L-NMMA as a nitric oxide synthetase inhibitor. DMSO and 100 nM insulin were used as controls. Values are represented as means ± SE (n=3). Different letters indicate significant differences between the groups (p < 0.05; Tukey HSD test).
Many reports have demonstrated that soy protein and its major component, βCG, have the potential to prevent and treat hyperglycemia and diabetes mellitus (Cederroth, et al., 2008; Lu, et al., 2008; Yang, et al., 2008; Tachibana, et al., 2014). We previously reported that consumption of βCG improves postprandial hyperglycemia by promoting GLUT4 translocation to the plasma membrane in skeletal muscle of a rat model of diabetes (Tachibana, et al., 2014). This suggests that βCG may act directly and/or indirectly in GLUT4 translocation. In this study, we found that treatment with βCG peptides promoted glucose uptake activity and GLUT4 translocation in skeletal muscle cells (Figures 2 and 3). A number of bioactive peptides derived from the enzymatic digestion of food proteins exhibit various physiological functions, including antihypertensive (Matoba et al., 1999; Zhao, et al., 2008) and cholesterol-lowering (Yamauchi, et al., 2003) activities. Yamada et al. (2011) showed that soymorphin-5, a pentapeptide derived from the β-subunit of βCG, improves glucose and lipid metabolism in KK-Ay mice, probably via activation of the adiponectin and PPARγ pathways, followed by increases in β-oxidation and energy expenditure. Mochizuki et al. (2009) showed that functional peptides derived from βCG hydrolysate suppress de novo triglyceride synthesis and the release of very low-density lipoprotein in hepatic HepG2 cells. Taken together, these results demonstrate that βCG peptides might promote glucose uptake accompanied by GLUT4 translocation in skeletal muscle cells. However, the concentration of βCG peptides used in this study was much higher than physiological concentrations, and βCG peptides revealed a wide molecular range. Thus, it is still unclear whether certain peptide(s) is incorporated into the muscle and contribute to the promotion of glucose uptake after intake of βCG. Further study is needed to clarify the active peptide of βCG on glucose metabolism in skeletal muscle cells.
In this study, we also found that βCG peptides promote GLUT4 translocation through the AMPK signaling pathway, but not the PI3K signaling pathway (Figures 4 and 5). This finding supports our previous report showing that consumption of βCG does not affect the phosphorylation of PI3K and Akt in skeletal muscle (Tachibana, et al., 2014). Further supporting an AMPK activation role for βCG peptides, L-NMMA did not affect βCG peptide-induced glucose uptake activity (Figure 6), suggesting that NO was not involved in βCG peptide-induced glucose uptake activity in L6 myotubes. AMPK activation is regulated by various biological factors, including LKB1 (Merry, et al., 2009). LKB1 activates AMPK and contributes to signal transduction from adiponectin receptor 1 (AR1) (Kadowaki and Yamauchi, 2005; Zhou, et al., 2009). Adiponectin is secreted from visceral fat pads, acting through the AR in the liver and muscles (Kadowaki and Yamauchi, 2005). Consumption of βCG increases serum adiponectin and induces AR1 mRNA expression and GLUT4 translocation in the skeletal muscle via activation of AMPK in spontaneously diabetic rats (Tachibana, et al., 2014). Given these results, βCG peptides may mimic adiponectin action or activate AR1 in L6 cells. An AR knockout muscle cell model will be a good tool to clarify this issue, which we are planning to use for future experiments.
In conclusion, βCG peptides increased glucose uptake by promoting GLUT4 translocation in L6 myotubes. As an underlying molecular mechanism, βCG peptides activate AMPK activity in skeletal muscle cells. Further studies are needed to clarify the exact mechanism after the identification of the active peptide(s). Our findings in this study provide an explanation for the preventive effects of insulin resistance and hyperglycemia after intake of βCG.
