Green tea extract with polyethylene glycol-3350

Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-013-0869-9

ORIGINAL ARTICLE

Green tea extract with polyethylene glycol-3350 reduces body weight and improves glucose tolerance in db/db and high-fat diet mice Jae-Hyung Park & Yoon Jung Choi & Yong Woon Kim & Sang Pyo Kim & Ho-Chan Cho & Shinbyoung Ahn & Ki-Cheor Bae & Seung-Soon Im & Jae-Hoon Bae & Dae-Kyu Song

Received: 29 December 2012 / Accepted: 2 April 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Green tea extract (GTE) is regarded to be effective against obesity and type 2 diabetes, but definitive evidences have not been proven. Based on the assumption that the gallated catechins (GCs) in GTE attenuate intestinal glucose and lipid absorption, while enhancing insulin resistance when GCs are present in the circulation through inhibiting cellular glucose uptake in various tissues, this study Electronic supplementary material The online version of this article (doi:10.1007/s00210-013-0869-9) contains supplementary material, which is available to authorized users. J.-H. Park : H.-C. Cho : K.-C. Bae : S.-S. Im : J.-H. Bae : D.-K. Song (*) Department of Physiology and Endocrinology, Keimyung University School of Medicine, 1095 Dalgubeoldae-Ro, Dalseo-Gu, Daegu 704-701, South Korea e-mail: [email protected] D.-K. Song e-mail: [email protected] S. P. Kim Department of Pathology, Keimyung University School of Medicine, 1095 Dalgubeoldae-Ro, Dalseo-Gu, Daegu 704-701, South Korea Y. J. Choi : Y. W. Kim Department of Physiology, Yeongnam University School of Medicine, 317-1, Daemyung-Dong, Nam-Gu, Daegu 705-717, South Korea S. Ahn GCB R&D and Clinical Trial Center Co., Ltd., 906-5 Eeu-Dong, Youngdong-Gu, Suwon, Gyeonggi-do, South Korea

attempted to block the intestinal absorption of GCs and prolong their residence time in the lumen. We then observed whether GTE containing the nonabsorbable GCs could ameliorate body weight (BW) gain and glucose intolerance in db/db and high-fat diet mice. Inhibition of the intestinal absorption of GCs was accomplished by co-administering the nontoxic polymer polyethylene glycol-3350 (PEG). C57BLKS/J db/db and high-fat diet C57BL/6 mice were treated for 4 weeks with drugs as follows: GTE, PEG, GTE + PEG, voglibose, or pioglitazone. GTE mixed with meals did not have any ameliorating effects on BW gain and glucose intolerance. However, the administration of GTE plus PEG significantly reduced BW gain, insulin resistance, and glucose intolerance, without affecting food intake and appetite. The effect was comparable to the effects of an α-glucosidase inhibitor and a peroxisome proliferator-activated receptor-γ/α agonist. These results indicate that prolonging the action of GCs of GTE in the intestinal lumen and blocking their entry into the circulation may allow GTE to be used as a prevention and treatment for both obesity and obesity-induced type 2 diabetes. Keywords Obesity . Diabetes . Green tea extract . Polyethylene glycol . Glucose uptake . Lipid absorption

Introduction Obesity can be induced by overeating, hypoactivity, and genetic factors. In general, individuals living in developing and developed countries consume high-calorie diets that provide more calories than they need, leading to body weight (BW) gain. There are strong positive associations

Naunyn-Schmiedeberg's Arch Pharmacol

among BW gain, insulin resistance, and type 2 diabetes (Mokdad et al. 2001; Rossner 2002). Especially, in individuals with a hereditary susceptibility to type 2 diabetes, obesity and/or insulin resistance may tend to cause dysfunction of beta cells (Perley and Kipnis 1966; Polonsky et al. 1988; Kahn et al. 1993; Kahn 2001). Therefore, it is generally accepted that reducing obesity could help prevent the development of type 2 diabetes. Green tea (leaves of Camellia sinensis, Theaceae) extract (GTE) is regarded as an herbal remedy for obesity and type 2 diabetes (Benelli et al. 2002; Weisburger and Chung 2002). When present in the intestinal lumen after oral ingestion, the gallated catechins (GCs) of GTE, such as epicatechin-3-gallate (ECG) and epigallocatechin-3-gallate (EGCG), have been shown to inhibit intestinal glucose uptake by inhibiting the type 1 sodium-dependent glucose transporter (SGLT1) (Kobayashi et al. 2000). In addition, GCs have also been shown to inhibit the intestinal absorption of lipids (Raederstorff et al. 2003). Nevertheless, the debate over whether GTE or its major component EGCG can prevent or treat human type 2 diabetes has not been settled (Anderson and Polansky 2002; Naftalin et al. 2003; Fukino et al. 2005). It is interesting that EGCG has not yet been adapted as a therapeutic drug for type 2 diabetes, whereas α-glucosidase inhibitors to block the cleavage of disaccharides into monosaccharides have already been used for type 2 diabetes. In a previous study, we suggested that GCs, at the doses that are normally ingestible by humans as daily green tea intake, can acutely reduce postprandial blood glucose levels primarily through their ability to inhibit the gastrointestinal SGLT1, whereas the circulating GCs increase blood glucose levels and insulin resistance by inhibiting cellular glucose transporters (GLUTs) in various tissues including skeletal muscle and adipose tissues (Park et al. 2009). The blood concentrations of GCs are known to peak at approximately 1 h after oral ingestion and remain at half the initial blood concentrations for at least 3 h (Yang et al. 1998; Chow et al. 2001; Lee et al. 2002). In addition, we found that higher concentrations of EGCG (>10 μM) are needed to decrease the number of adipocytes, which is probably through intracellular generation of reactive oxygen species (Sung et al. 2010). Animals and humans have been reported to suffer from significant adverse effects at such a high blood concentration of EGCG (Naftalin et al. 2003; Yin et al. 2009), indicating the doses being clinically inapplicable against obesity and type 2 diabetes. This study was designed to elucidate whether GTE ingestion, at the doses that are well tolerable by humans (Chow et al. 2001; Van Amelsvoort et al. 2001; Isbrucker et al. 2006), can ameliorate BW gain and glucose intolerance in db/db and high-fat diet mice. In addition, we used polyethylene glycol with molecular weight of 3,350 Da (PEG) to block the intestinal absorption of GCs, which is

nontoxic (Wilkinson 1971; Attar et al. 1999) and selectively binds the GCs of GTE (Park et al. 2009) to form a complex. Our previous report revealed that PEG co-administered with GTE blocked the impact of GTE on postprandial blood glucose levels (Park et al. 2009). The effects of GTE alone and GTE + PEG were compared with those of an αglucosidase inhibitor (voglibose) and a peroxisome proliferator-activated receptor-γ/α agonist (pioglitazone), which are currently used for type 2 diabetes as therapeutic drugs.

Materials and methods Chemicals and diets PEG was obtained from Kukjeon Pharma (Seoul, South Korea). Green tea leaves (BOSUNG SEIJAK) were purchased from Bosung Green Tea Co. (Jeonnam, South Korea). EGCG was purchased from Sigma-Aldrich (St. Louis, MO, USA). Voglibose and pioglitazone were kind gifts from CJ Cheiljedang Pharma (Seoul, South Korea) and Lilly Korea (Seoul, South Korea), respectively. Commercial normal chow diet (10 % fat, 70 % carbohydrate, 20 % protein), normal chow diet containing PEG (1 g PEG/kg diet), normal chow diet containing GTE (10 g GTE/kg diet), normal chow diet containing GTE plus PEG (10 g GTE/kg and 1 g PEG/kg diet), normal chow diet containing voglibose (0.014 g voglibose/kg diet), and normal chow diet containing pioglitazone (0.1 g pioglitazone/kg diet) were prepared by Hyochang Science (Seoul, South Korea). High-fat diet was composed of protein, carbohydrate, and fat (20, 20, and 60 %, respectively, of total calories) supplemented with vitamins (1 %) and minerals (3.5 %), while the caloric composition of “AIN93G”, control, was 20, 64, and 16 % (protein, carbohydrate, and fat, respectively). High-fat or control diet containing PEG (1 g PEG/kg diet), high-fat or control diet containing GTE (10 g GTE/kg diet), high-fat or control diet containing GTE plus PEG (10 g GTE/kg and 1 g PEG/kg diet), high-fat or control diet containing voglibose (0.014 g voglibose/kg diet), and high-fat or control diet containing pioglitazone (0.1 g pioglitazone/kg diet) were prepared by Hyochang Science. Preparation of GTE For animal studies, 20 g of green tea leaves was added to 1,000 ml of ultrapure water. After being stirred for 5 min at 80 °C, the tea leaves were removed by filtration using filter paper (Advantec 2 filter paper, Hyundai micro Co., Seoul, South Korea) under reduced pressure. The extract was dried by lyophilization. A total of 3 g of GTE was harvested, in which EGCG, ECG, EGC,


and EC accounted for 100, 53, 56, and 31 mg/g GTE, respectively. Nuclear magnetic resonance spectroscopy of the EGCG-PEG complex Nuclear magnetic resonance (NMR) spectroscopy was used to investigate molecular interactions between EGCG, the representative of GCs, and PEG within the complex. 1H NMR and 13C NMR spectra were measured using a JEOL JNM-AL 300 (300 MHz) spectrometer. Sample of the EGCG-PEG (w/w, 1:1) complex was prepared by dissolving the mixture in D2O/DMSO-d6 (1:1, Sigma-Aldrich) [concentration of 0.25 % (w/v)]. Caco-2 cell culture Human colon adenocarcinoma Caco-2 cells were purchased from the Korean Cell Line Bank (Seoul, South Korea). Cells were grown and incubated in Dulbecco’s Modified Eagle Medium (DMEM) with a high glucose concentration (4.5 g/l) supplemented with 10 % fetal calf serum, 1 % nonessential amino acids, penicillin (50 mU/ml), and streptomycin (50 mg/ml). The medium was changed daily. Uptake of EGCG by Caco-2 cells Caco-2 cell were seeded onto 24-well size BD Falcon Cell Culture Inserts (BD Biosciences, San Jose, CA, USA) at 6× 105 cells/cm2 (200,000 cells/insert) in a serum-free medium consisting of DMEM and Mito+ Serum Extender (BD Biosciences). The inserts contain a track-etched polyethylene terephthalate 1 μm microporous membrane. The seeding medium was replaced 24 h after cell seeding with differentiation medium (Entero-STIM Differentiation Medium, BD Biosciences). After 48 h, apical to basal permeability assays were performed. Briefly, Caco-2 cells were incubated in Hank’s Balanced Salt Solution (HBSS) for 30 min. Then, the cells were treated with 100 μM EGCG or 100 μM EGCG plus 20 μM PEG for 180 min. The level of EGCG in the basolateral fluid compartment was determined by HPLC analysis.

indicated concentrations for 0, 60, 120, or 180 min. The level of glucose uptake was determined by adding a mixture of D-[6-3H] glucose (1 μCi; final concentration, 0.1 μM) and 20 mM unlabeled glucose. After a 10-min incubation, the reaction was stopped by three quick washes with icecold phosphate-buffered saline (PBS). The cells were then lysed in PBS containing 0.2 M NaOH, and glucose uptake was assessed by scintillation counting. Western blot analysis Caco-2 cells were washed twice in ice-cold PBS, and total cellular proteins were extracted in lysis buffer [10 mM Tris– Cl (pH 7.4), 130 mM NaCl, 5 % (v/v) Triton X-100, 5 mM EDTA, 200 nM aprotinin, 20 mM leupeptin, 50 mM phenanthroline, and 280 mM benzamidine HCl] for 20 min at 4 °C. The protein concentrations were measured using the Bio-Rad (Hercules, CA, USA) protein assay. Cellular lysates were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were then probed with antiSGLT1 (Abcam, Cambridge, UK) and anti-β-actin (Sigma). The immunoreactive bands were visualized with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK). The experiments were repeated at least three times. Oral glucose tolerance test An oral glucose tolerance test (OGTT) was performed on normal mice. On test days, water, PEG, EGCG, GTE, or GTE + PEG was administered orally to fasted (12 h) mice. PEG (90 mg/kg), EGCG (90 mg/kg), GTE (900 mg/kg; 90 mg EGCG/kg), and GTE + PEG (900 mg/kg and 90 mg/kg, respectively) were suspended in water and given orally prior to the administration of glucose. Either immediately or 60 min later, 2 g/kg glucose was given orally. The blood glucose levels were measured in tail blood samples collected at 0, 15, 30, 60, 90, and 120 min after the glucose treatment. Blood glucose levels were measured using the Glucocard Test Strip II (Arkray Inc., Kyoto, Japan).

Glucose uptake measurement in Caco-2 cells Animals and treatments Caco-2 cells were seeded in 24-well culture plates at a density of 2×105 cells with serum-free medium consisting of DMEM and Mito+ Serum Extender (BD Biosciences). The seeding medium was replaced 24 h after cell seeding with differentiation medium. After 48 h, glucose uptake assays were performed. Briefly, Caco-2 cells were incubated in HBSS, a glucose-free medium, for 2 h. Then, the cells were treated with PEG, EGCG, or EGCG plus PEG at the

To determine the effects of GTE plus PEG on obese and type 2 diabetic mice, C57BLKS/J db/db mice (male, 9 weeks old, 30.0–40.0 g, blood glucose level 200–400 mg/dl) and age-matched control nondiabetic heterozygous mice (male, 20.0–23.9 g, blood glucose level 110–185 mg/dl) were purchased from Jung-Ang Experimental Animals (Seoul, South Korea). Mice were allowed to acclimate for 1 week


on chow and water. From 10 weeks of age, db/db mice were provided with a semisynthetic, normal chow diet containing PEG, GTE, GTE plus PEG, voglibose, or pioglitazone for 4 weeks. In addition, 4-week-old C57BL/6 mice were treated with a high-fat diet or control diet for 8 weeks. After 8 weeks of high-fat or control diet, PEG, GTE, GTE plus PEG, voglibose, or pioglitazone was provided with the diet for 4 weeks. The food consumption of individual rats was checked every day based on the difference between the amount of chow supplies each day and the amount of chow remaining. The BW was measured every 7 days using an electronic balance. All mice had free access to food and water. The animals were housed under a daily 12 h light/12 h dark cycle. Animals were treated as approved by the Keimyung University Institutional Ethics Committee, Daegu, South Korea.

were based on NCBI’s nucleotide database and were designed using the Primer Express program (Applied Biosystems): mouse POMC (forward, 5′-ACC TCA CCA CGG AAA GCA A-3′; reverse, 5′-CGG GGA TTT TCA GTA AAG G-3′) and mouse NPY (forward, 5′-TAT CCC TGC TCG TGT GTT TG-3′; reverse, 5′-GTT CTG GGG GCA TTT TCT G-3′). Liver and pancreas histopathology Embedded liver and pancreas tissue blocks were cut into 6-μm sections and stained with hematoxylin and eosin. A diagnosis of fatty liver was made based on the presence of macro- or microvesicular fat in >5 % of the hepatocytes in a given slide. Tissue lipid determination

Intraperitoneal glucose tolerance test An intraperitoneal glucose tolerance test (IPGTT) was performed after each 4-week drug treatment on control and diabetic mice. On test days, the animals were fasted for 12 h and then given an intraperitoneal (i.p.) injection of glucose (500 mg/kg). The blood glucose levels were measured in tail blood samples collected at 0, 15, 30, 60, 90, and 120 min after the glucose treatment. Collection of blood and internal organ samples At the conclusion of the study, a 500-μl blood sample was collected from the orbital venous plexus. After blood sampling, visceral adipose tissues (perirenal, retroperitoneal and epididymal depots), the liver, the pancreas, and the hypothalamus were dissected out. The hypothalamus tissues were immediately frozen in liquid nitrogen and stored at −80 °C until measurement of mRNA levels by real-time RT-PCR. Real-time RT-PCR analysis Each whole mouse hypothalamus was homogenized in TRI reagent (Sigma-Aldrich) using an Ultra-Turrax T25 (Staufel, Germany). RNA was reverse transcribed to cDNA from 1 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed using the Real-Time PCR 7500 system and Power SYBR Green PCR master mix (Applied Biosystems), according to the manufacturer’s instructions. The expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The reactions were incubated at 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s, 55 °C for 20 s, and 72 °C for 35 s. Primers for mouse proopiomelanocortin (POMC), neuropeptide Y (NPY), and GAPDH

Samples of liver were homogenized in 0.25 % sucrose containing 1 mM EDTA. Lipids were extracted using chloroform/methanol (2:1, v/v) and evaporated in a SpeedVac, and the pellets were dissolved in 5 % fatty acid-free bovine serum albumin dissolved in water. The amount of protein in the homogenate was assayed using a protein assay reagent (Bio-Rad, Hercules, CA, USA) to normalize the amount of extracted lipids. The tissue triglyceride (TG) levels were determined using kits from Roche Diagnostics (Seoul, South Korea). Biochemical analyses of plasma samples The plasma adiponectin levels were determined with the Mouse Adiponectin/Acrp30 Immunoassay (R&D Systems, Minneapolis, MN, USA). The plasma retinol-binding protein 4 (RBP4) levels were determined with the Mouse RBP4 Immunoassay (Adipogen, Incheon, South Korea). Fasting glucose and insulin were measured using commercial kits (SpinReact, Gerona, Spain; Millipore, Billerica, MA, USA, respectively). Calculation of HOMA-insulin resistance The HOMA-insulin resistance (IR) was calculated using the final blood glucose and insulin levels in food-deprived mice. This method is widely used to estimate insulin resistance in humans and animal models (Konrad et al. 2007; Mlinar et al. 2007). Statistical analysis The results are expressed as means±SEM. SPSS (version 14.0; SPSS Inc., Chicago, IL, USA) was used for the statistical analyses. The AUC was calculated using MicroCal


Origin software (version 7.0; Northampton, MA, USA). For comparisons of more than two groups, significance was tested using ANOVA with the Bonferroni correction to address the relatively small numbers of samples. P values less than 0.05 were considered significant.

Results Effect of GTE on glucose tolerance in normal mice Orally administered GTE resulted in lower blood glucose levels during the OGTT than the control when glucose was given simultaneously (Fig. 1a, b). This result is consistent with the previously reported results for rats and humans (Park et al. 2009). However, the results of the OGTT Fig. 1 Effect of GTE + PEG during the OGTT in normal mice. Changes in blood glucose levels (a, b) during the OGTT (2 g glucose/kg) when performed immediately after administration of water, PEG, GTE, or GTE + PEG. Changes in blood glucose levels (c, d) during the OGTT when performed 1 h after the drug administration. Changes in blood glucose levels (e, f) during the OGTT (2 g glucose/ kg) when performed immediately after administration of water, EGCG (90 mg/kg), or GTE (900 mg/ kg; 90 mg EGCG/kg). Mice were fasted overnight before the experiments. AUC is depicted in b, d, f as the percentage of the control value.*P