Green tea extract with polyethylene glycol-3350 reduces body weight and improves glucose tolerance in db/db and high-fat diet mice
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 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.
KeywordsObesity Diabetes Green tea extract Polyethylene glycol Glucose uptake Lipid absorption
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 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 α-glucosidase 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.
Glucose uptake measurement in Caco-2 cells
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 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 ice-cold 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 anti-SGLT1 (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).
Animals and treatments
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.
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 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
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).
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.
Effect of GTE on glucose tolerance in normal mice
PEG inhibits intestinal absorption of EGCG in vitro
EGCG inhibits glucose uptake in Caco-2 cells and the effect is prolonged by PEG
GTE + PEG reduces BW gain independent of food intake in obese–diabetic mice
GTE + PEG reduces glucose intolerance and insulin resistance
GTE + PEG ameliorates liver and islet pathologies
The intestinal glucose uptake on the apical side is mainly mediated by SGLT1, which is the main target for the beneficial effects of orally ingested GTE (Kobayashi et al. 2000; Shimizu et al. 2000). GCs are known to play the primary role in this inhibition (Kobayashi et al. 2000). The present results clearly show that administration of GTE is more effective than the administration of EGCG alone. Although ECG is one of the important green tea GCs, the result may support previous observations showing that other ingredients of GTE may protect GCs from degradation (Osada et al. 2001; Chacko et al. 2010). The inhibitory effect of GCs on cellular glucose uptake has also been observed in mouse adipocytes (Nomura et al. 2008), rat adipocytes (Strobel et al. 2005), and human erythrocytes (Naftalin et al. 2003). Our previous study (Park et al. 2009) revealed that the glucose uptake of tested cells including hepatocytes and myocytes was hindered by EGCG. Therefore, it was hypothesized that GCs could inhibit the action of most plasmalemmal glucose transporters, including SGLTs and Na+-independent GLUTs (Johnston et al. 2005). Although the precise mechanism is unknown, the effects of GCs on glucose transporters are likely to be the result of steric hindrance caused by incorporation into the membrane with the subsequent disruption of the surrounding lipid bilayer, as shown previously using transfected Xenopus oocytes (Hossain et al. 2002). An additional effect of GTE in the gastrointestinal lumen may be the inhibition of cholesterol absorption (Raederstorff et al. 2003). GCs appear to be responsible for the action, primarily through the inhibition of mixed micelle formation (Raederstorff et al. 2003).
One problem may be that certain proportions of the ingested GCs are timely absorbed into the circulation (Park et al. 2009). The time-dependent decrease in the effect of EGCG on glucose uptake in Caco-2 cells may be due to both its degradation and its uptake by cells, as EGCG has been shown to be taken up by cells (Hong et al. 2002). Despite the possibility of luminal degradation, the OGTT performed 1 h after oral GTE ingestion clearly indicates that more elevated blood glucose levels than the control may be due to circulating GCs to be absorbed. The intestinal effect of GCs would not be shown at the case of GTE alone because animals have empty stomach when GTE is ingested. The effect of circulating GCs may be detrimental, particularly in the postprandial period (Arakawa et al. 2001). Thus, it is plausible that the effects of GCs in the circulation explain why GTE alone is not effective in db/db and high-fat diet mice, as shown in humans (Fukino et al. 2005). We speculate that pro-diabetic effect of oral administration of GTE might be shown in an in vivo long-term study if GTE can be supplied to animals between meals, not with meals as the present study. This may be because of beta cell overload by GCs-induced defects in postprandial blood glucose excursion. The effects of GCs in both the circulation and intestines on inhibiting cellular glucose uptake were measurable after the ingestion of a range of doses achievable by daily green tea consumption (Sung et al. 2010). Therefore, it likely has clinical significance. Fortunately, at the concentrations at which GCs affect glucose absorption in Caco-2 cells, GCs do not inhibit peptide and amino acid transporters (Kobayashi et al. 2000).
The lack of effect on appetite by the oral administration of GTE is in good agreement with the previous finding that observed for mice maintained on a high-fat, high-sucrose diet supplemented with EGCG (Klaus et al. 2005). However, the mice maintained on this diet had reduced BW gain compared with non-EGCG-supplemented mice, in contrast to the present results. This discrepancy may be attributable to the difference in the concentrations of EGCG used: our regimen used approximately ten times less EGCG than the previous experiment (Klaus et al. 2005) (0.1 % EGCG vs. 0.94 % EGCG in supplied diets). There is a report showing that a parenteral administration of EGCG (80–90 mg/kg, i.p. once a day) reduces appetite and BW of normal and obese Zucker rats within 7 days (Kao et al. 2000), although their interest was mainly focused on EGCG-mediated prostate cancer therapy. The lower food intake and BW loss were shown to be related with EGCG-induced hypotrophy of male sexual organs and reduced blood testosterone levels (Kao et al. 2000; Naftalin et al. 2003), supporting that EGCG inhibits glucose uptake of androgen-producing cells (Naftalin et al. 2003). In that experiment, the reduced appetite and BW were also observed even in normal rats, which render limitation to a circulating EGCG-based therapy at least for obesity and type 2 diabetes.
To block the effects of GCs in the circulation and prolong the effects of GCs in the intestinal lumen, we used the resin PEG, which is known to be only minimally absorbable by the intestinal epithelium and has negligible toxicity (Wilkinson 1971; Attar et al. 1999), to selectively inhibit the intestinal absorption of GCs (Park et al. 2009). The present study used NMR and confirms that interactions exist between EGCG and PEG. Remarkably, the co-administration of GTE + PEG, but not GTE alone, in the diet for 4 weeks significantly ameliorated BW gain and glucose intolerance in db/db and high-fat diet mice; the effects was comparable to the effects of voglibose and pioglitazone. This result may suggest that, only with the intestinal effect of GTE to inhibit glucose and lipid absorption, it is possible to attenuate the progression of obesity-induced type 2 diabetes. As noted, the beneficial effect of GTE + PEG on glucose tolerance appears to result from lower BW gain and decreased body fat mass, a similar mechanism to the α-glucosidase inhibitor voglibose (Negishi et al. 2008). Accordingly, the regimen alleviated changes in fatty liver, pancreatic islet size, and the levels of serum adipokines (Abbasi et al. 2004; Yang et al. 2005; Graham and Kahn 2007). Thiazolidinediones are well known to reduce liver fat content (Leclercq et al. 2007). In the present study, the result is not statistically significant. However, it is shown that the mice supplied with 0.01 % pioglitazone-containing diet for 4 weeks had slightly lower hepatic TG contents than the control obese mice. Therefore, it is suggested that a higher dose or longer exposure would give statistically significant results for pioglitazone as strong as GTE and PEG.
In conclusion, dietary GTE + PEG alleviated BW gain and insulin resistance in db/db and high-fat diet mice, thus ameliorating glucose intolerance. PEG effectively blocks the absorption of GCs into the circulation and prolongs the intestinal effects of GCs. Therefore, the GTE + PEG complex may be a preventative and therapeutic tool for obesity and obesity-related type 2 diabetes without much concern about systemic side effects. Further study remains to determine whether it is effective in human obesity and type 2 diabetes.
This work was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (grant number 110135-3). Miss Sun-Joo Kim in Keimyung University School of Medicine provided valued technical assistance and care of the animals.
Conflict of interest
The authors declare that they have no conflict of interest.
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