Berberine improves glucose metabolism through induction of glycolysis
Berberine, a botanical alkaloid used to control blood glucose in type 2 diabetes in China, has recently been reported to activate AMPK. However, it is not clear how AMPK is activated by berberine. In this study, activity and action mechanism of berberine were investigated in vivo and in vitro. In dietary obese rats, berberine increased insulin sensitivity after 5-wk administration. Fasting insulin and HOMA-IR were decreased by 46 and 48%, respectively, in the rats. In cell lines including 3T3-L1 adipocytes, L6 myotubes, C2C12 myotubes, and H4IIE hepatocytes, berberine was found to increase glucose consumption, 2-deoxyglucose uptake, and to a less degree 3-O-methylglucose (3-OMG) uptake independently of insulin. The insulin-induced glucose uptake was enhanced by berberine in the absence of change in IRS-1 (Ser307/312), Akt, p70 S6, and ERK phosphorylation. AMPK phosphorylation was increased by berberine at 0.5 h, and the increase remained for ≥16 h. Aerobic and anaerobic respiration were determined to understand the mechanism of berberine action. The long-lasting phosphorylation of AMPK was associated with persistent elevation in AMP/ATP ratio and reduction in oxygen consumption. An increase in glycolysis was observed with a rise in lactic acid production. Berberine exhibited no cytotoxicity, and it protected plasma membrane in L6 myotubes in the cell culture. These results suggest that berberine enhances glucose metabolism by stimulation of glycolysis, which is related to inhibition of glucose oxidation in mitochondria. Berberine-induced AMPK activation is likely a consequence of mitochondria inhibition that increases the AMP/ATP ratio.
berberine is a botanical alkaloid in the roots and bark of several plants, including Coptis chinensis French, an ancient Chinese herb that has been used to treat diabetes for thousands of years in China. Berberine is the main active compound of the herb. In addition to its metabolic activities, berberine has well-established antimicrobial activities in the control of infection by bacteria, viruses, fungi, protozoans, and helminthes (8, 14). It is an over-the-counter drug for the treatment of gastrointestinal infections in China. In 1988, the hypoglycemic effect of berberine was found when berberine was used to treat diarrhea in diabetic patients in China (13). Since then, berberine has been used as an antihyperglycemic agent by many physicians in China. There are many clinical reports about the hypoglycemic action of berberine in Chinese literature.
Regarding the mechanism of berberine action, we found that berberine increased glucose metabolism in cultured cells, and this activity was comparable to that observed for metformin in 2002 (20). This activity was confirmed later in other studies, and AMPK was proposed to mediate the metabolic activities of berberine (2, 3, 9, 22). In 2006, it was reported that berberine was able to activate AMPK for inhibition of lipid synthesis in human hepatocytes (2). Berberine was reported to reduce body weight and improve glucose metabolism in animal models of metabolic syndrome (9). Berberine was found to induce phosphorylation of AMPK in 3T3-L1 adipocytes and L6 myotubes, and AMPK was proposed to explain the insulin-sensitizing effect of berberine (9). Activation of the AMPK pathway by berberine was also observed by two other groups (3, 22). Although AMPK is activated by berberine, the metabolic conditions that lead to this effect require further investigation. Berberine was shown to inhibit the citric acid cycle in isolated mitochondria (1, 11, 12). However, it is not clear whether this activity can be observed in living cells for AMPK activation.
In this study, the metabolic activity of berberine was examined in diabetic rats, and the action mechanism was investigated in cellular models. The result suggests that berberine stimulates glucose metabolism through induction of glycolysis. Activation of AMPK by berberine is related to an increase in the AMP/ATP ratio. Inhibition of glucose oxidation in mitochondria may contribute to the AMP/ATP ratio increase and AMPK activation.
RESEARCH DESIGN AND METHODS
DMEM, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin solution, 2-deoxy-d-glucose, 3-O-methyl-d-glucopyranose, and cytochalasin B were purchased from Sigma Chemicals (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Atalanta Biologicals (Lawrenceville, GA). Glucose color reagent was purchased from Raichem (San Diego, CA). 2-Deoxy-d-[3H]glucose and 3-O-d-[methyl-14C]glucose were purchased from PerkinElmer (Boston, MA). Antibodies to phospho-Akt (Thr308) and phospho-p70 S6 kinase (Thr389) were purchased from Cell Signaling Technology (Danvers, MA). Anti-goat IgG [horseradish peroxidase (HRP) conjugated], antibodies to Akt1/2, GLUT4, and phospho-IRS-1 (Tyr632) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to phospho-IRS-1 (Ser307) was purchased from Upstate (Charlottesville, VA). Antibodies to α-tubulin and GLUT1 were obtained from Abcam (Cambridge, MA). HRP-conjugated anti-rabbit IgG or anti-mouse IgG were purchased from GE Healthcare UK (Buckinghamshire, UK). PVDF membrane for immunoblot was purchased from Bio-Rad (Hercules, CA). Protein assay kit was purchased from Pierce (Rockford, IL). Berberine for animal experiments was from the Materia Medica Factory of Meitan County, Guizhou Province, China. Berberine for in vitro experiments was from Sigma Chemicals (St. Louis, MO).
Male Wistar rats (Experimental Animals Center of Chinese Academic, Shanghai, China), 2∼3 mo old and ∼240 g body wt, were housed individually in wire cages in a temperature-controlled room (22 ± 1°C) on a 12:12-h light-dark cycle. The animals were fed a high-fat diet (HFD; lard, 59% of calories) with free access to food and water. The animal protocol was approved by the institutional review board. After 6 mo on HFD, the animals were divided into control (n = 6) and berberine-treated (n = 6) groups. Berberine was administrated at 125 mg/kg twice a day at 1000 and 2200 by gastric gavage for 5 wk. Control rats were gavaged with an equal volume of vehicle (distilled water). After 5-wk treatment, an intraperitoneal glucose tolerance test (IPGTT) was conducted between 0900 and 1100 on 10-h fasted animals. Fasting blood was first collected by lacerating the tail vein before glucose was given at 2 g/kg body wt (40% glucose solution). Then blood samples were collected at 2 h. Fasting insulin was detected using a LINCO Rat Insulin RIA Kit (St. Charles, MO). The homeostasis model assessment (HOMA) method was used to determine insulin resistance (HOMA-IR) (10): HOMA-IR = fasting insulin (μU/ml) × fasting glucose (mmol/l)/22.5.
Mouse fibroblast 3T3-L1 preadipocytes, L6 rat skeletal myoblasts, C2C12 mouse myoblasts, and rat hepatoma cell line H4IIE were purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM culture medium supplemented with 10% FBS, 4 mmol/l glutamine, and 50 mg/l gentamicin in an atmosphere of 5% CO2 at 37°C. For adipogenesis, 3T3-L1 preadipocytes were grown to confluence in a 100-mm plate and then treated with an adipogenic cocktail (5 μg/ml insulin, 0.5 mmol/l IBMX, and 10 μmol/l dexamethasone) for 4 days. This was followed by incubation in insulin-supplemented medium for an additional 3 days. The normal medium was used at day 7 to maintain the adipocytes (6). For differentiation of myotubes, L6 cells were cultured in DMEM with 10% FBS for 24 h. The cells were then maintained in 2% FBS medium (α-MEM) for 6 days to induce differentiation. The medium was changed every 48 h (15). For C2C12 cells, DMEM supplemented with 2% horse serum was used to induce differentiation. The inducing process was similar to that of L6 cells (16).
The cells were cultured in 96-well tissue culture plates. When experiments were conducted, the normal culture medium was replaced by DMEM supplemented with 0.25% bovine serum albumin (BSA). Then, the cells were treated with berberine and/or insulin (final concentration 100 nmol/l) for 24 h. The glucose concentration in medium was determined by the glucose oxidase method. The glucose of the wells with cells was subtracted from the glucose of the blank wells to obtain the amount of glucose consumption (20).
3T3-L1 preadipocytes, L6, or C2C12 myoblasts were differentiated into mature adipocytes or myotubes in a 12-well plate. For 2-deoxyglucose (or 3-OMG) uptake, after berberine treatment in serum-free DMEM with 0.25% BSA for 16 h, the cells were treated with insulin (200 nmol/l) for 20 min at 37°C in PBS (1 ml/well). After a washing in PBS, the cells were incubated in 1 ml of PBS containing 0.1 mmol/l 2-deoxy-d-glucose (or 3-O-methyl-d-glucose) and 1 μCi/ml 2-deoxy-d-[3H]glucose (or 3-O-d-[methyl-14C]glucose) for 5 min. Then, the cells were washed twice in ice-cold PBS and lysed in 0.4 ml of 1% sodium dodecyl sulfate. [3H]glucose (or [14C]glucose) content was determined in 4 ml of scintillant using a Beckman LS6500 scintillation counter. Nonspecific glucose uptake is measured in the presence of 20 μmol/l cytochalasin B and subtracted from the total uptake to get specific glucose uptake (6, 18).
Cells were treated with berberine in serum-free medium with 0.25% BSA. In some experiments, insulin (200 nmol/l) was added 20 min before cell collection. The whole cell lysate was made by sonication in lysis buffer (1% Triton X-100, 50 mmol/l KCl, 25 mmol/l HEPES, pH 7.8, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 125 μmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l sodium orthovanadate). The protein (100 μg) was boiled for 5 min, resolved in 7% mini-SDS-PAGE for 90 min at 100 V, and blotted on the polyvinylidene difluoride membrane (Bio-Rad). After being preblotted in milk buffer for 30 min, the membrane was blotted with the first antibody for 2–12 h and the secondary antibody for 30 min. The HRP-conjugated secondary antibodies were used with chemiluminescence reagent (PerkinElmer Life Sciences) for generation of the light signal. To detect multiple signals from one membrane, the membrane was treated with a stripping buffer (0.5 mol/l NaOH) for 20 min after each cycle of blotting to remove the bound antibody. All of the experiments were conducted three times (5). ImageJ 1.37V was used to quantify the Western signals.
LDH cytotoxicity assay.
3T3-L1 adipocytes and L6 myotubes were cultured in 24-well plates. After berberine treatment for 24 h in serum-free DMEM supplemented with 0.25% BSA, lactate dehydragenase (LDH) concentration in the medium was detected with the LDH-Cytotoxicity Assay Kit II (BioVision, Mountain View, CA).
Oxygen consumption was performed in BD Oxygen Biosensor Systems (BD Biosciences, Bedford, MA). After differentiation, the 3T3-L1 adipocytes or L6 myotubes were trypsinized and plated to a plate embedded with an oxygen-sensitive dye in DMEM culture medium supplemented with 10% FBS. After 6 h, berberine and/or insulin (final concentration 100 nmol/l) was added to the medium. Fluorescence reading was taken at different times. The units of oxygen consumption were normalized relative fluorescence units (NRFU), which were obtained by dividing the fluorescence values of cells by the value of the blank wells.
Adenine nucleotide contents assay.
3T3-L1 adipocytes and L6 myotubes were cultured in six-well plates. They were treated with 5 μmol/l berberine for 0.5 or 16 h in serum-free medium with 0.25% BSA. Then ATP and AMP contents of the cells were measured using high-performance liquid chromatography (HPLC) as described by Wynants et al. (17). In brief, the cells were lysed in 0.6 N HClO4 and neutralized with 1 N KHCO3. HPLC analysis was developed on an HPLC system (Waters Delta 600, Waters, Milford, MA) consisting of a solvent delivery pump unit (Waters 600), an autosampler (Waters 717 Plus), and a UV-Vis diode array detector (Waters 2996 Photodiode Array Detector, 190–800 nm). The system was computer controlled and analyzed with the Empower software system (Waters Delta 600). Separation was carried out using an Atlantis T3 column (5.0 μm, 4.6 × 150 mm ID; Waters) with guard column (4.6 × 20 mm ID; Waters). The mobile phase consisted of an aqueous buffer containing 0.15 M phosphoric acid (10 ml of 85% H3PO4/liter) adjusted to pH 6.00 with ammonium hydroxide (∼8 ml; A) and a mixture of acetonitrile and methanol (50:50, vol/vol; B). The optimal mobile phase was set as below: 0–5 min, 100% A; 5–25 min, gradient to A/B = 90:10; the flow rate was set as 0.7 ml/min, and the wavelength was set at 259 nm. ATP and AMP appeared at 9.6 and 18.1 min, respectively.
The cells were cultured in a 24-well plate and treated with berberine and/or insulin in serum-free DMEM supplemented with 0.25% BSA. The lactate concentration in the medium was detected with a lactate assay kit from Biomedical Research Service Center, University of Buffalo (Buffalo, NY).
Data are presented as means ± SE. All experiments in cells were performed at least in triplicate. When a single comparison was performed, the significance of the differences between means was analyzed by Student’s t-test. When multiple comparisons were performed, the significance was analyzed by one-way ANOVA (SPSS 12.0). When a significant effect was found, differences between means were determined by Fisher’s least significant difference post hoc test. The P level was set at 0.05.
Berberine improved insulin sensitivity in dietary obese rats.
In this study, dietary obese rats were used as an animal model of insulin resistance. At 6 mo on the HFD, blood glucose was significantly elevated in the Wistar rats in fasting and fed conditions. With 5-wk treatment by berberine, both fasting blood glucose (FBG) and postprandial blood glucose (PBG) were significantly reduced in the dietary obese rats (Fig. 1A). FBG was reduced from 5.74 to 5.39 mmol/l (P = 0.035). PBG was reduced from 10.4 to 7.6 mmol/l (P = 0.039), which is close to the normal glucose level. Fasting insulin level was decreased by 46% (P = 0.045; Fig. 1B). The insulin sensitivity was increased significantly by berberine, as indicated by a 48% reduction (P = 0.036; Fig. 1C) in HOMA-IR….