Effects of unaltered and bioconverted mulberry leaf extracts on cellular glucose uptake and antidiabetic action in animals

Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), RPMI-1640 medium, phosphate-buffered saline (PBS), and trypsin-EDTA were obtained from Gibco BRL (Grand Island, NE, USA). Penicillin/streptomycin was obtained from Thermo Fisher Scientific (Rockford, IL, USA). Insulin, NA, STZ, glimepiride, metformin, D-(+)-glucose, 2-deoxy-D-glucose, 3-isobutyl-1-methylxanthine (IBMX), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), NaCl, KCl, MgSO₄, CaCl₂, KH₂PO₄, and NaHCO₃ were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide was obtained from VWR Life Science (Radnor, PA, USA). 2-Deoxy-[³H]-glucose was obtained from PerkinElmer Life Sciences (Boston, MA, USA). Rosiglitazone was obtained from Masung & Co., Ltd. (Seoul, Korea). All other chemicals were of analytical grade. Trans-Caffeic acid and syringaldehyde were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Other standard compounds (morin 3-O-β-D-glucopyranoside, moracin M 3′-O-β-glucopyranoside, and astragalin) were provided by Professor Young Ho Kim in College of Pharmacy at Chungnam National University (Daejeon, South Korea). All standards were higher than 95% of purity. Methanol, ethyl acetate and acetonitrile were purchased with HPLC grade from Honeywell Burdick & Jackson (Muskegon, MI, USA). Formic acid (HPLC grade) was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Other chemicals used were GR (Guaranteed Reagent) grade. Ultrapure water was manufactured by a Milli-Q water purification system (ShinHan, Daejeon, South Korea). Anti-phospho-AKT serine 473 (S473) and AKT antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-β-actin and anti-rabbit antibodies were purchased from AbFrontier (Geumcheon, Seoul, Korea).

Dried mulberry leaves were purchased in September 2015 at the Naemome Dah herb company in Ulsan, Korea. The herb was taxonomically identified by Professor Young Ho Kim who is an expert in the area of Natural Product Chemistry. A voucher specimen (CNU 16004) was deposited at the Herbarium of College of Pharmacy, Chungnam National University, Daejeon, Korea. Dried leaves (100 g) were reconstituted in about 20 times its weight of purified water, pressure-extracted at 121 °C for 3 h, and then filtered. The filtered extract was concentrated using a vacuum concentrator (Rovator R-200; BÜCHI Korea Inc., Seoul, Korea) and 16 g powder was obtained using a freeze dryer. Solutions of the resulting MLE powder were created at a concentration of 300 or 600 mg/kg in normal saline for the animal experiments.

Water MLE was adjusted to pH 4.5, the enzyme Viscozyme L (Novozyme, Copenhagen, Denmark) was added at a concentration of 1% dried leaf, and the fermentation process was conducted at 45 °C for 15 h, after which the enzyme activity was terminated at 90 °C for 30 min. The solution was filtered, concentrated using a vacuum evaporator, and freeze-dried to obtain 19 g powder. The resulting BMLE powder was used at concentrations of 300 or 600 mg/kg in normal saline for the animal experiments.

The marker compounds in BMLE and MLE used in this study were analyzed by Prof. Jong Seong Kang in College of Pharmacy at Chungnam National University (Daejeon, South Korea). This method is under review for Analytical Journal.

Five-week-old C57BL/6 mice (18–23 g) were purchased from OrientBio (Seongnam, Korea). The mice were acclimatized for 1 week at 22 ± 2 °C and 40 ± 5% humidity, and allowed free access to a normal- or high-fat diet (60% kcal fat, Research Diets, Inc., New Brunswick, NJ, USA) and drinking water. All animal handling was performed according to the guidelines of the Committee for Ethical Usage of Experimental Animals at Chungnam National University (CNU-00773; Daejeon, South Korea). In all experiments using animals, an inhalational anesthetic, isoflurane, was used to alleviate animal pain, and they were euthanized by cervical dislocation after completing experiments.

After fasting for 16 h, mice fed on high-fat diet for 8 weeks were intraperitoneally (IP) injected by NA (240 mg/kg) 15 min before administration of STZ (100 mg/kg, IP) to induce obese diabetic condition as described previously [28, 29, 33]. To confirm whether a diabetic condition was established in animals, oral glucose tolerance test (OGTT) was performed and the results were compared with type 1 diabetic mice induced by administration of STZ (100 mg/kg) alone. These results showed that STZ-NA-induced diabetic mice had different conditions from STZ-induced ones (Additional file 1).

The experimental mice were randomly divided into eight groups containing seven mice in each group as follows:

One week before the end of the experiment, all mice in the eight groups were fasted for 16 h. Glucose (2 g/kg, PO) was administered 30 min after the administration of the vehicle or agents, and blood was collected from the tail vein of each animal at 0, 15, 30, 45, 60, and 120 min to measure blood glucose with a blood glucose meter (Accu-Chek; Roche Diagnostics Korea Co., Ltd., Seoul, Korea). The OGTT data were plotted using GraphPad, and significant differences among groups were evaluated by measuring the AUC of each line graph.

The ITT followed the same protocol as the OGTT, except for the subcutaneous administration of insulin (1 U/kg) 30 min after glucose administration (2 g/kg, PO). Blood was collected from the tail vein of each animal after 0, 30, 45, 60, and 120 min, and blood glucose was measured. The data interpretation was the same as that described for the OGTT.

Mice were fasted for 16 h before the final administration of vehicle or agent. Mice were anesthetized 30 min after the final administration via isoflurane inhalation (2–3% isoflurane with oxygen supply; Isoflurane 100, JW PHARMA, Daejeon, Korea). After laparotomy, about 0.7–1 mL blood was collected through the abdominal artery in the EDTA-coated tube. Plasma was separated by centrifugation at 4000 rpm for 15 min, and plasma insulin was measured with a mouse insulin ELISA kit (Cat. AKRIN-011 T; Shivayagi, Gunma, Japan) according to the manufacturer’s instructions. The amount of insulin released from HIT-T15 cells into the media was measured as previously described [34]. HIT-T15 pancreatic β-cells seeded on a 24-well at a density of 2 × 10⁵ cells/well were treated with RPMI-1640 medium supplemented with 10% FBS and 11.1 mM glucose and were cultured for 2 days. After washing with Krebs-Ringer bicarbonate-HEPES (KRBH) buffer (119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl₂·2H₂O, 1.19 mM MgCl₂·6H₂O, 1.19 mM KH₂PO₄, 25 mM NaHCO₃, and 7 mM HEPES; pH 7.4) without glucose, cells were incubated in KRBH buffer with 0.3% bovine serum albumin for 30 min, which was exchanged with KRBH buffer containing 11.1 mM glucose, and then treated with the prescribed agent for 1 h. Insulin levels released from cells into the KRBH buffer were measured with an insulin assay kit as described above. After lysing cells with 150 μL 0.5 N NaOH, the protein concentration in cell lysate was measured. Standard curves were established using the standard materials included in the ELISA kit, and plasma insulin levels (ng/mL) and insulin secretion (ng insulin/mg protein) were measured using a microplate reader (Infinite M200 PRO; Tecan Group Ltd., Zürich, Switzerland) at 450 nm.

HbA_1C levels in the whole blood of mice were measured using a mouse HbA_1C Assay Kit (Cat. 80,310; Crystal Chem, Elk Grove Village, IL, USA) according to the manufacturer’s instructions. After establishing a standard curve, HbA_1C levels were determined using a microplate reader at 700 nm [35].

The HOMA-IR and QUICKI indices were calculated as follows:

C2C12 myotubes and 3 T3-L1 adipocytes were cultured and differentiated as previously described [36, 37]. Briefly, C2C12 myoblasts were cultured at 37 °C under a 5% CO₂ atmosphere in DMEM containing 10% FBS. Confluent myoblasts were designated as day 0, and DMEM containing 1% FBS was replaced daily for 6 days to differentiate C2C12 myotubes. 3 T3-L1 preadipocytes were cultured at 37 °C in a 5% CO₂ atmosphere in DMEM containing 10% calf serum for 2 days, and confluent 3 T3-L1 cells were designated as day 0. Cells at day 0 were treated with 10% FBS, 0.5 mM IBMX, 1 μM dexamethasone, and 1 μg/mL insulin for 3 days to induce differentiation. After 3 days of differentiation, cells were maintained in DMEM containing 10% FBS and 1 μg/mL insulin for 2 days. Then the cell medium was replaced with DMEM containing 10% FBS, which was replaced every 2 days until day 8. HIT-T15 pancreatic β-cells were cultured at 37 °C in a 5% CO₂ atmosphere in RPMI-1640 culture medium containing 10% FBS, 0.8 mM glucose, 100 U/mL penicillin, and 100 μg/mL streptomycin, and were subcultured for 3–5 days [34].

3 T3-L1 adipocytes or C2C12 myotubes grown in DMEM with 1% FBS were treated with Krebs-Ringer phosphate-HEPES buffer (136 mM NaCl, 4.7 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 10 mM phosphate buffer, and 10 mM HEPES; pH 7.4). Cells were treated with medium alone (basic glucose uptake) or with 100 nM insulin (insulin-stimulated glucose uptake) for 30 min. Glucose uptake was initiated with adding 100 μM 2-deoxy-D-glucose together with 2-deoxy-[³H]-glucose (0.1 μCi/mL in 3 T3-L1 adipocytes or 0.5 μCi/mL in C2C12 myotubes) per well. After 10 min, the cells were washed three times with cold PBS and lysed using 0.5 M NaOH and 0.1% sodium dodecyl sulfate. The radioactivity was determined using liquid scintillation counting with a β-counter (1600TR; BioSurplus, Inc., San Diego, CA, USA) and normalized according to the total protein level [36, 37].

Total proteins in differentiated C2C12 cells were extracted using RIPA buffer and quantified with BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Proteins in the sample buffer were boiled for 10 min and resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (ATTO Corp., Tokyo, Japan) at 4 °C for 80 min at 120 mA. After blocking with TBS-T (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) containing 5% bovine serum albumin (BSA) for 1 h, the membranes were then incubated with a 1:1000 dilution of primary antibodies targeting the following; phospho-AKT (S473), AKT, and β-actin. After removing primary antibodies, the blots were washed three times in TBS-T, incubated with anti-rabbit antibody diluted 1:2000 in TBS containing 5% BSA for 5 h at 4 °C and then washed five times in TBS-T. Antibody-bound proteins were detected using enhanced chemiluminescence (AbFrontier, Seoul, Korea) and imaging systems (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instruction. The expression level of each protein was normalized to β-actin and the intensities of the bands were quantified using Quantity One (Bio-Rad, Hercules, CA, USA) [38, 39].

HPLC analysis showed two major marker compounds in MLE, compounds 1 (trans-caffeic acid, Fig. 1a) and 2 (syringaldehyde, Fig. 1b), and the quantities of these two compounds in BMLE were increased by 184 and 280%, respectively (Table 1), indicating that bioconversion process increases the quantity of marker compounds compared with unaltered MLE. To confirm the abilities of two marker compounds to uptake glucose into cells, two compounds with variable concentrations (0.5–5 μM) were applied to C2C12 myotubes. Both trans-caffeic acid and syringaldehyde significantly increased insulin-stimulated glucose uptake in a concentration-dependent manner (Fig. 1c). These results indicate that the more concentration of these compounds provides the greater ability of glucose uptake.

We compared the abilities of BMLE and MLE to increase glucose uptake in differentiated C2C12 myotubes and 3 T3-L1 adipocytes and insulin secretion from HIT-T15 pancreatic β-cells. Insulin-stimulated glucose uptake significantly increased in C2C12 myotubes treated with 5 or 10 μg/mL BMLE (Fig. 2a). BMLE treatment increased insulin-stimulated glucose uptake from 140 to 152% at a concentration of 5 μg/mL and to 159.1% at 10 μg/mL. By contrast, MLE showed no significant increase in glucose uptake. Neither MLE nor BMLE affected basal glucose uptake. Similarly, 20 μg/mL BMLE significantly increased insulin-stimulated glucose uptake in 3 T3-L1 adipocytes (Fig. 2b). Moreover, the ability of BMLE (20 μg/mL) to increase insulin-stimulated glucose uptake (from 400 to 449.3%) was significantly greater than that of MLE (20 μg/mL; from 400 to 420.7%). As a positive control, rosiglitazone (10 μM) significantly increased insulin-stimulated glucose uptake up to 487.9%. Although 20 μg/mL BMLE significantly increased insulin secretion in HIT-T15 pancreatic β-cells, MLE did not show any stimulating effect (Fig. 2c). As a positive control, glimepiride (1 μg/mL) showed the greatest efficacy in stimulating insulin secretion in this model. These results indicate that bioconversion improved the ability of MLE to increase insulin-stimulated glucose uptake in skeletal muscle cells and adipocytes, as well as insulin secretion in pancreatic β-cells. BMLE and MLE at greater doses (20 or 50 μg/mL) than those used in the experiments showed no cytotoxicity for 1 or 2 h in these cells (In vitro test of the cytotoxicity; Additional file 2).

Insulin-mediated AKT phosphorylation induces GLUT4 translocation from cytosol to membrane, enhancing glucose uptake [40, 41]. To confirm this linkage, the effect of two marker compounds, MLE and BMLE on AKT phosphorylation was measured in C2C12 myotubes (Fig. 3). Trans-caffeic acid significantly increased AKT phosphorylation (Fig. 3a). Syringaldehyde did not show any statistical significant increase at given concentrations, but exert an increasing tendency. As expected, both BMLE and MLE significantly increased AKT phosphorylation (Fig. 3b). Notably, BMLE had a greater effect on AKT phosphorylation than MLE at the same concentration (10 μg/mL). These results suggest that bioconversion increase in amount of trans-caffeic acid, which causes the greater effect than MLE on AKT phosphorylation-induced glucose uptake.

To compare the abilities of BMLE and MLE to improve glucose tolerance, obese diabetic mice were established by co-administering STZ and NA as described previously [28, 29]. The body weights of both BMLE- and MLE-treated mice fed a high-fat diet decreased (Fig. 4a). As a positive control, metformin (300 mg/kg, PO) treatment also resulted in a decrease in body weight. However, fasting blood glucose levels measured once a week were significantly decreased in both the BMLE (600 mg/kg, PO)- and metformin-treated groups (Fig. 4b). Note that the quantities of two marker compounds in BMLE dosage is greater than those in MLE dosage (Table 2).

The oral glucose tolerance test (OGTT) was performed after 6 weeks of treatment, and the results were plotted with a line graph, using the area under the curve (AUC) of each line to compare the differences among treatments (Fig. 5a). Both the BMLE- and MLE-treated groups showed significantly decreased blood glucose levels compared to the control (untreated obese diabetic mice fed a high-fat diet). The blood glucose levels in the BMLE-treated group were lower than those in the MLE-treated group at the same dose. The insulin tolerance test (ITT) was performed after 7 weeks of treatment, and significant changes among groups were compared based on the AUC (Fig. 5b). After injecting insulin, blood glucose levels in both the BMLE- and MLE-treated groups were significantly lower than those in the control group. Moreover, the AUC was significantly lower in the 600 mg/kg BMLE-treated group than in the 600 mg/kg MLE-treated group. In both tests, the AUCs of metformin were similar to those of the high concentration-treated BMLE group. These results indicate that BMLE had a better ability to regulate blood glucose levels in the obese diabetic animal model than MLE.

After the final BMLE and MLE treatment in mice, HbA_1C blood and plasma insulin levels were measured. The HbA_1C levels in both the BMLE- and MLE-treated groups were significantly lower than those in the control group (Fig. 6a). As expected, the HbA_1C levels in the BMLE-treated group were significantly lower than those of the MLE-treated group at the same dosage. Plasma insulin levels were significantly increased in only the group treated with 600 mg/kg BMLE (Fig. 6b). Next, the homeostasis model assessment of insulin resistance (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) values were compared among the groups (for the calculations, see the Materials and Methods). The HOMA-IR value in the 600 mg/kg BMLE-treated group was significantly lower than that in the MLE group, but was similar to that in the metformin-treated group (Fig. 7a). Moreover, the QUICKI value of the 600 mg/kg BMLE-treated group was higher than that of the MLE group (Fig. 7b). These results indicate that BMLE showed a better capacity than MLE to decrease blood HbA_1C levels, increase insulin secretion, and improve insulin resistance and insulin sensitivity.