Hypoglycemic Activity and the Mechanisms of Lycium Bark Extract in db/db Mice

Abstract

The extract of Lycium bark (LBE), which is the root bark of Lycium chinense, has long been used in China for hypertension, inflammation, and diabetes. LBE has been reported to ameliorate hyperglycemia in mice with alloxan-induced type 1 diabetes, but evidence on the effect of LBE in diabetes had not been enough. Therefore, we investigated the effects of LBE on type 2 diabetes using db/db mice. Nine-week-old male db/db mice were orally administered LBE (425 mg/kg) for 10 weeks. Blood samples were collected under anesthesia for the determination of blood glucose and insulin levels. The blood glucose level was increased in the control group and was unchanged in the LBE group. The blood insulin level was increased in both groups within 4 weeks, but it decreased in the control group and was maintained at a relatively high level in the LBE group thereafter. Furthermore, LBE increased the glucose uptake, which was measured using C2C12 myotubes, in a concentration-dependent manner, independent of the addition of a phosphatidylinositol 3-kinase inhibitor (i.e., LY294002) and an AMP-activated kinase inhibitor (i.e., dorsomorphin). And LBE increased the mRNA expression of glucose transporter (GLUT) 1. These results suggested that LBE decreased the blood glucose level by additive effect such as improvement of the insulin secretion, promoting activity of glucose uptake. These findings suggested that LBE administration can be a novel therapeutic approach for type 2 diabetes.

INTRODUCTION

In Japan, the number of patients with diabetes continues to increase every year. According to the National Health and Nutrition Survey in Japan in 2016, the number of persons in whom diabetes is strongly suspected was estimated to be approximately 10 million, that is, 1 of 10 people may have diabetes. Diabetes mellitus is a problem not only by itself, but it also increases the risk for cardiovascular disease and causes complications, such as neuropathy, retinopathy, and kidney disease. Therefore, prevention of diabetes is important.

Diabetes is classified according to cause, as type 1 or type 2. Approximately 90% of Japanese diabetic patients are diagnosed with type 2 diabetes, which is characterized by insulin resistance, and insulin hyposecretion from pancreatic β cells.¹⁾ The major pathophysiology of type 2 diabetes has been attributed to lifestyle, such as overeating, lack of exercise, and obesity, in addition to genetic influence. In particular, obesity initiates insulin resistance, which reduces glucose uptake in muscles and adipose tissues, and leads to persistent hyperglycemia.²⁾ To improve hyperglycemia and normalize the blood glucose levels, pancreatic β cells increase the quantity of insulin secretion. However, when such a situation continues, the pancreas becomes exhausted and induces insulin hyposecretion, which causes hyperglycemia and eventual development of type 2 diabetes. In general, glycemic control has been thought to be important in the prevention of diabetes.

For a long time, the fruit, leaf, and bark of the plant Lycium chinense or L. barbarum (Solanaceae) had been used for medicinal purposes. Lycium bark is a component of the Kampo formula, such as Jiinshihoto or Seishinrenshiin. Recently, Lycium bark has been reported to exert hypotensive, hepatoprotective, and anti-inflammatory activities.^3,4) In “Shennong Bencaojing (神農本草経),” L. chinense was reported to have an effect on “wasting and thirsting (消渇)” disease, but its medicinal part was not described. The major characteristics of “wasting and thirsting (消渇)” include excessive thirst, hunger, and urination, and emaciated body and can be regarded as similar to those of diabetes. In previous studies on the constituents of these plants, kukoamine, betaine and polysaccharides have been isolated.³⁾L. barbarum polysaccharide has been reported to have an effect on a type 2 diabetes model.^5,6) Moreover, Lycium bark has been reported to exert hypoglycemic activity in an alloxan model of type 1 diabetes, but its effects on type 2 diabetes have not been investigated.⁷⁾ In the present study, we used db/db mouse as a rodent model of type 2 diabetes to evaluate the effects of the Lycium bark extract (LBE). In addition, we evaluated the mechanism of LBE using skeletal muscle cells.

MATERIALS AND METHODS

Preparation of Plant Extracts

The root bark of L. chinense Mill. was purchased in China, and a voucher specimen (03C1C2) was placed in our laboratory. Extracts were obtained by placing 200 g of small dried pieces of the root bark of L. chinense Mill. in 10-fold boiling water for 30 min. After filtration, the supernatant was concentrated to finally obtain 48 g (i.e., 24% yield) of LBE. Using the LBE, the presence of scopoletin, which is one of the components of the Lycium bark, was confirmed by TLC⁸⁾ (Supplementary Fig. S1).

Animals

Eight-week-old male BKS.Cg-Dock7^m +/+ Lepr^db/J (db/db) mice and C57BL/6J mice were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). The mice were housed under a 12 h light and 12 h dark cycle in a room with controlled temperature (24 ± 2°C) and humidity (55 ± 10%). The mice were allowed food and water ad libitum. All experimental procedures conformed to the Regulations for the Management of Laboratory Animals of Matsuura Yakugyo Co., Ltd. and were approved by the Institutional Animal Care and Use Committee of Matsuura Yakugyo Co., Ltd.

Evaluation of the Type 2 Diabetes Model Mouse

Following a week of acclimatization, the db/db mice were randomly divided into two groups, according to body weight and blood glucose level: the control group, and the LBE group (n = 8). The normal (C57BL/6J mice) and control groups were given water ad libitum, whereas the LBE group was orally administered with LBE (425 mg/kg) dissolved in drinking water for 10 weeks. The dose of 425 mg/kg/d was calculated from the previous report.⁷⁾ Blood samples were collected from the retroorbital venous plexus under anesthesia for determination of the blood glucose and insulin levels, using an automated biochemical analyzer (Spotchem™ SP-4410, Arkray Co., Kyoto, Japan) and the Lbis Insulin-mouse-U enzyme-linked immunosorbent assay (ELISA) Kit, which was purchased from FUJIFILM Wako Shibayagi Corporation (Gunma, Japan).

Measurement of Nitric Oxide (NO) Production

NO production was determined by measuring the nitrite (NO₂) concentration. RAW264.7 macrophages (The European Collection of Authenticated Cell Cultures, Parton Down, U.K.) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, MO, U.S.A.) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin in 5% CO₂ at 37°C. The cells were seeded in 96-well plates at 1 × 10⁵ cells/well and were incubated for 24 h. LBE (213–1064 µg/mL) was added; the cells were stimulated by the addition of interferon-γ (50 U/mL) and lipopolysaccharide (LPS, 0.5 µg/mL) after 30 min, and 3 h, respectively. At 18 h after stimulation with LPS, the amount of NO production in the medium was detected using the Griess reagent (1% sulfanilamide, and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid); each supernatant was mixed with the same volume of the Griess reagent. The absorbance at 540 nm was measured with a plate reader (MULTISKAN SPECTRUM; ThermoFisher Scientific, Waltham, MA, U.S.A.), and nitrite concentration was determined using a dilution of sodium nitrite as the standard. The positive control was 1 µM of N^G-monomethyl-L-arginine (L-NMMA) (DOJINDO LABORATORIES, Kumamoto, Japan).

C2C12 Culture

Mouse C2C12 cells (the American Type Culture Collection, Manassas, VA, U.S.A.) were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in 5% CO₂ at 37°C. For differentiation of myoblasts into myotubes, the cells were seeded in 12-well plates at 1.5 × 10⁴ cells/well. After reaching 90% confluence, the cells were cultured in DMEM containing 2% horse serum (HS), and 100 nM insulin (Sigma-Aldrich) for three days. Thereafter, the cells were cultured with DMEM containing 2% HS, without insulin, for 3 more days and had differentiated into myotubes. Then, the medium was changed to DMEM containing LBE, and myotubes were cultured for 24 h. In addition, 2 mM of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 1 µM of insulin were used as positive control. At the time of the experiment using an inhibitor, DMEM containing 7 µM of LY294002 (FUJIFILM Wako Pure Chemical Corporation) or 7.5 µM of dorsomorphin (AdooQ Bioscience, Irvine, CA, U.S.A.) was added 30 min before the addition of LBE or the positive control.

2-Deoxyglucose Uptake Assay

The cells were washed twice with Krebs–Ringer phosphate buffer (KRPH), which comprised 20 mM of N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 5 mM of KH₂PO₄, 1 mM of MgSO₄.7H₂O, 1 mM of CaCl₂.2H₂O, 136 mM of NaCl, and 4.7 mM of KCl and had a pH of 7.4. Then, the cells were incubated for 30 min at 37°C in the KRPH buffer containing 0.1 mM of 2-deoxyglucose (2-DG). After incubation, the cells were washed twice with ice-cold phosphate buffered saline (PBS), and were lysed with 10 mM Tris–HCl (pH 8.0). The amount of glucose in the lysate was measured using a 2-DG Uptake Measurement Kit (Cosmo Bio Co., Ltd., Tokyo, Japan).

mRNA Extraction and Real-Time PCR

The total RNA from the C2C12 myotubes, which were cultured with the samples for 24 h, was extracted using RNAiso Plus (TaKaRa Bio Inc., Shiga, Japan). RNA was converted into cDNA by the RT method using the ReverTra Ace^® (TOYOBO CO., LTD., Osaka, Japan). The relative quantification of the mRNA of glucose transporters (GLUTs) 1 and 4 was measured by a comparative CT method (ΔΔCT) of real-time PCR, using the Applied Biosystems StepOne™ Real-time PCR System with Fast SYBR Green Master Mix (Thermo Fisher Scientific). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as an internal control. The nucleotide primers for GLUT1, GLUT4, and GAPDH were designed by the primer 3 software, and were blasted in the NCBI PubMed primer blast software. The forward and reverse primer sequences were: 5′-TGC TCC TCG TGC TCT TCT TC-3′, 5′-CTC CTC GGG TGT CTT GTC A-3′ for GLUT1; 5′-CGG GCA AAG GAA CAC AAT AG-3′, 5′-TGG AGG GGA ACA AGA AAG TG-3′ for GLUT4; and 5′-GGA TGC AGG GAT GAT GTT CT-3′, 5′-ACC CAG AAG ACT GTG GAT GG-3′ for GAPDH. Real-time PCR was performed under the following conditions: 20 s at 95°C, 40 cycles within 1 s at 95°C, and within 20 s at 60°C.

Statistical Analysis

The results were expressed as mean ± standard deviation (S.D.). Statistical analysis was performed by Dunnett’s test and Bonferroni correction. A p-value of <0.05 with Dunnett’s test, or a p-value of <0.0033 with Bonferroni correction was considered statistically significant.

RESULTS

Hypoglycemic Activity of LBE in db/db Mice

The db/db mice showed progressive body weight gain, while body weight and the food intake were not significantly different between the control and LBE group (Fig. S2). When the exam started at the age of 9 weeks, the blood glucose and insulin levels of the db/db mice were significantly higher than those of normal mice. The control db/db mice showed progressive blood glucose elevation for 4 weeks and maintained this high level until the end of the examination. LBE treatment markedly prevented elevations and maintained the baseline levels of the blood glucose at all times (Fig. 1A), and decreased water consumption in relation to blood glucose (Fig. S2). The blood insulin level of the control db/db mice were elevated, similar to the blood glucose levels, for 4 weeks, but it decreased to half thereafter. On the other hand, LBE treatment prevented hyposecretion and maintained a high level of insulin (Fig. 1B).

Fig. 1. Effect of LBE on Hyperglycemia in db/db Mice

The normal (C57BL/6J mice) and control (db/db mice) groups were given water, whereas the LBE group (db/db mice) was orally administered with LBE (425 mg/kg) dissolved in drinking water for 10 weeks. Each group was evaluated for (A) fasting blood glucose level every 2 weeks and (B) fasting blood insulin level every 4 weeks. Data are expressed as mean ± S.D. (n = 8). * p < 0.05, ** p < 0.01 compared with the control by the Dunnett’s test.

Inhibitory Action of NO Production

Chronic inflammation of adipose tissues causes disorders in glucose metabolism.⁹⁾ Therefore, we examined the anti-inflammatory actions of LBE. The NO production by the RAW264.7 cells stimulated by interferon-γ and LPS was inhibited by the addition of L-NMMA as a positive control. Under these conditions, LBE showed inhibitory actions on NO production in a concentration-dependent manner (Fig. 2).

Fig. 2. Effect of LBE on IFN-γ/LPS-Induced NO Production in RAW264.7 Cells

RAW264.7 cells were treated with LBE (213–1064 µg/mL) or 1 µM of L-NMMA. After 30 min, the RAW264.7 cells were stimulated, as described in the Materials and Methods section. Following the stimulation with LPS for 18 h, the amount of NO in the supernatant was measured by the Griess method. Data are expressed as mean ± S.D. (n = 4). ** p < 0.01 compared with the control by Dunnett’s test.

Promotion of Glucose Uptake in Skeletal Muscles

Because the skeletal muscle is an organ that performs maximum uptake of glucose, we examined the action of LBE on skeletal muscles using C2C12 myotubes. In C2C12 myotubes, AICAR, and insulin, as the positive control, significantly stimulated glucose uptake; LBE also increased glucose uptake in a concentration-dependent manner (Fig. 3). Then, we used inhibitors to investigate the mechanism involved in LBE-induced glucose uptake. Addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin inhibited insulin or AICAR-mediated glucose uptake but did not significantly affect LBE-induced glucose uptake (Fig. 4).

Fig. 3. Effect of LBE on Glucose Uptake in C2C12 Myotubes

C2C12 myotubes were treated with LBE (250–1000 µg/mL), 2 mM of AICAR, or 1 µM of insulin for 24 h. Then, the myotubes were incubated with 0.1 mM of 2-DG for 30 min, and the amount of 2-DG incorporated into the cells was measured using the assay kit. Data are expressed as mean ± S.D. (n = 4). * p < 0.05, ** p < 0.01 compared with the control by Dunnett’s test.

Fig. 4. Influence of Inhibitors on Glucose Uptake of LBE

Following the treatment with (A) 7 µM LY294002 or (B) 7.5 µM dorsomorphin for 30 min, C2C12 myotubes were treated with 1000 µg/mL of LBE. Then, myotubes were incubated with 0.1 mM 2-DG for 30 min, and the amount of 2-DG incorporated into cells were measured using the assay kit. Data are expressed as mean ± S.D. (n = 4). ** p < 0.0007 (=0.01/15) compared with the corresponding value by Bonferroni correction.

Glucose Transporter mRNA Expression

In skeletal muscles, GLUT1 and GLUT4 are mainly involved in glucose uptake; therefore, we investigated the GLUT mRNA expression using C2C12 myotubes. The GLUT4 mRNA level increased after insulin and AICAR treatment, but it was not affected by LBE. On the other hand, LBE treatment increased the GLUT1 mRNA level in a concentration-dependent manner (Fig. 5).

Fig. 5. Effect of LBE on GLUT mRNA Expression

C2C12 myotubes were treated with LBE (250–1000 µg/mL), 2 mM of AICAR, or 1 µM of insulin for 24 h. The mRNA levels of GLUT4 and GLUT1 were analyzed by real-time PCR. The mRNA levels of the control were assigned values of 1. Data are expressed as mean ± S.D. (n = 4). * p < 0.05, ** p < 0.01 compared with the control by the Dunnett’s test.

DISCUSSION

In this study, we investigated the effect of the Lycium bark on type 2 diabetes using db/db mice. The fasting blood glucose level of db/db mice is about two times as high as that of normal mice. As the examination progressed, the blood glucose level of the control db/db mice increased, and reached the upper limit at 4 weeks, and this high blood glucose level was sustained until the end of the examination. Similarly, the blood insulin level was high for 4 weeks, although it decreased to half thereafter. We inferred that the cause of this hyposecretion was glucotoxicity, based on the report that chronic exposure to a hyperglycemic state by overeating led to pancreatic β cell exhaustion and decline in insulin biosynthesis/secretion.¹⁰⁾ In this study, LBE treatment inhibited an increase and maintained the baseline blood glucose level, and it did not lead to insulin hyposecretion. These results indicated that LBE can improve hyperglycemia and further protect pancreatic function indirectly.

In addition to insulin hyposecretion, insulin resistance is also a characteristic of type 2 diabetes. In obese individuals, the adipose tissues are infiltrated with macrophages that secrete inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6, and release free fatty acids.^9,11,12) Because these inflammatory mediators can act on skeletal muscles, insulin resistance can ensue. Therefore, we thought that insulin resistance may be prevented by improving the inflammatory state. Indeed, we found that LBE inhibited NO production in a concentration-dependent manner; therefore, LBE may have anti-inflammatory actions. In the future, studies that use adipocytokine as an index of the improvement effect of LBE on insulin resistance will be needed.

Next, we investigated the mechanism of action of LBE in inducing hypoglycemia. Insulin or exercise plays an important role in glycemic control; the blood glucose level decreases by promoting glucose uptake from blood into a skeletal muscle or adipose tissue. Notably, in healthy individuals, the skeletal muscle is an organ that accounts for approximately 40% of the body weight, and consumes approximately 70% of the glucose supply of the entire body.¹³⁾ Therefore, we thought that the skeletal muscle is an important tissue for glycemic control. Indeed, our experiments showed that LBE promoted glucose uptake into skeletal muscles in a concentration-dependent manner, and suggested that it participated in improvement of hyperglycemia.

Glucose uptake in skeletal muscles occurs by the translocation of the skeletal muscle-specific GLUT4 from an intracellular pool to the cell surface. Insulin and muscle contraction during exercise stimulate GLUT4 translocation by different signal pathways. Insulin binds its receptor and activates the PI3K/Akt pathway, whereas exercise consumes ATP and activates the AMPK pathway.^14,15) The results of our present study showed that the promoting activity of LBE on glucose uptake was not affected by the PI3K and AMPK inhibitors. In addition, LBE did not affect GLUT4 mRNA expression, but it significantly upregulated GLUT1 mRNA, which is expressed in various tissues universally.^16,17) These results suggested that GLUT1 upregulation may be involved in the LBE-induced glucose uptake in skeletal muscles. But it is predicted to the involvement of other mechanisms because GLUT1 mRNA upregulation was little in the concentration of 250–500 µg/mL. As for PI3K/Akt pathway, PI3K phosphorylate Akt via phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-triphosphate (PIP3). Following Akt phosphorylate tre-2/USP6, BUB2, cdc16 domain family, member 4 (TBC1D4), phosphorylation of TBC1D4 increased 14-3-3 binding to this protein, which is proposed to inhibit Rab-GTPase-activating protein (GAP) activity. In consequence, activation of Rab induces GLUT4 translocation.^18,19) Whereas AMPK phosphorylate both TBC1D1 and TBC1D4 in AMPK pathway, which inhibits Rab-GAP function.^18,19) From these things, it cannot deny the possibility of not only GLUT1 upregulation, but also GLUT 4 translocation via phosphorylation of Akt or TBC1D1/4, or via Rab function may be involved in glucose uptake of LBE. We need further detailed investigation to elucidate of this mechanism.

Some natural products possess antidiabetic activities. For example, Salacia reticulata was reported to prevent diabetes by inhibiting the activities of α-glucosidase, and lipase.^20–22) Moreover, the berberine contained in the Phellodendron bark and the Coptis root was reported to contribute to lowering the blood glucose level of type 2 diabetes patients.^23,24) Therefore, natural products have been proven to be clinically effective for diabetes treatment.

In conclusion, LBE showed improvement of insulin hyposecretion, anti-inflammatory effects and promoting activity of glucose uptake. It was suggested that LBE exhibit hypoglycemic activity via insulin-sensitizing action as a result of additive effect of these action mechanisms. These findings suggested that the LBE administration can be a novel therapeutic approach for type 2 diabetes.

Conflict of Interest

All authors are employees of Matsuura Yakugyo CO., LTD.

Supplementary Materials

The online version of this article contains supplementary materials.

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