Chemicals and diets
γ-PGA (MW: 2,000 KDa) was delivered from NUC Electronics Co. (Daegu, Korea). Green tea leaves (Bosung Seijak) were purchased from Bosung Green Tea Co. (Bosung, Korea). A normal commercial chow diet (NC; 10% fat, 70% carbohydrate, 20% protein), a normal chow diet containing γ-PGA (1 g γ-PGA/kg diet), a normal chow diet containing GTE (10 g GTE/kg diet), and a normal chow diet containing GTE+γ-PGA (10 g GTE/kg and 1 g γ-PGA/kg diet) were prepared by Hyochang Science (Seoul, Korea). Briefly, each ingredient was weighed and blended to homogeneity into appropriately powdered diet. The mixture was then formed into equal sized pellets and placed into a temperature- and humidity-controlled room to remove moisture.
Preparation of GTE
For animal studies, 20 g of green tea leaves was added to 1,000 mL of nanopure water. The tea leaves were stirred for 5 minutes at 80
and removed by filtration using filter paper (Advantec 2 filter paper; Hyundai Micro Co., Seoul, Korea) under reduced pressure. The extract was dried by lysophilization. A total of 3 g of GTE was harvested. The procedure was repeated until enough amount of GTE necessary for the experiments were harvested; the levels of EGCG, ECG, epigallocatechin, and epicatechin were 100, 53, 56, and 31 mg/g GTE, respectively. Periodically, the diet containing GTE was checked to confirm the concentrations of catechins exceeding 200 mg/g GTE.
Analysis of catechins from GTE with high performance liquid chromatography
High performance liquid chromatography (HPLC) analysis was conducted on a Waters Alliance 2,695 liquid chromatograph equipped with a model 2,487 dual absorbance detector (Waters Co., Milford, MA, USA). A Waters Symmetry C18 reversed-phase packing column (4.5×250 mm, 5 m) was used at 25
for separation throughout this study. Catechins were determined simultaneously at 235 nm. A gradient elution was performed by varying the proportion of solvent A (water-trifluoroacetic acid, 99.9:0.1 v/v) to solvent B (acetonitrile-trifluoroacetic acid, 99.9:0.1 v/v), with a flow rate of 1 mL/min. The mobile phase composition changed linearly from 9.5% to 14% solvent B in 10 minutes and then kept the same composition for 10 minutes. There was a linear increase in the proportion of solvent B, which reached 27.5% within 15 minutes. The mobile phase composition then returned to the initial conditions over a period of 5 minutes for the next run. All prepared solutions were filtered through 0.45 µm membranes (Sartorius, Maisemore, UK), and the mobile phase was degassed before injection into the HPLC.
Nuclear magnetic resonance spectroscopy of EGCG-γ-PGA complex
Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the molecular interactions between EGCG and γ-PGA. 1H NMR and 13C NMR spectra were measured on a JEOL JNM-AL 300 (300 MHz) spectrometer. The sample for EGCG-γ-PGA (w/w, 1:1) complex was prepared by dissolving it in D2O/DMSO-d6 (1:1; Sigma-Aldrich, St. Louis, MO, USA) (concentration of 0.25% [w/v]).
Animals and treatments
To determine the short-term effects of GTE+γ-PGA on type 2 diabetic mice, C57BLKS/J Leprdb/Leprdb mice (db/db, male, 9 weeks old, 30.0 to 35.0 g, BGLs 200 to 300 mg/dL) and age-matched control nondiabetic heterozygous mice (male, 18.0 to 22.0 g, BGLs 110 to 140 mg/dL) were purchased from Jung-Ang Experimental Animals (Seoul, Korea) and used as an obese diabetic mouse model. The mice were allowed to acclimate for 1 week on chow and water. Starting at 10 weeks of age, the mice were randomly divided into four groups of 10: control, γ-PGA, GTE, and GTE+γ-PGA. The mice were provided with a semisynthetic normal chow diet containing vehicle, γ-PGA, GTE, or GTE+γ-PGA for 4 weeks. The food consumption of individual mice was checked every day. BW was measured every 7 days using an electronic balance. All mice had free access to food and water and received their specified diet for 4 weeks. The animals were housed (3 to 4 per cage) under a daily cycle of 12 hours light and 12 hours darkness. The experiment conditions were approved by the Keimyung University Institutional Ethics Committee, Daegu, Korea, which supervises animal research.
Oral and intraperitoneal glucose tolerance test
Oral glucose tolerance test (OGTT) was performed on normal mice. The mice were randomly divided into six groups of seven each: control, GTE, GTE+γ-PGA, GTE+1/2γ-PGA, GTE+1/3γ-PGA, and GTE+1/4γ-PGA. On the test days, GTE (900 mg/kg containing 90 mg EGCG/kg) and γ-PGA (90, 45, 22.5, and 11.25 mg/kg) were suspended in water and administered orally to fasted (12 hours) mice. Ninety minutes later, 2 g/kg glucose was given orally. The BGLs were measured in tail blood samples collected at 0, 15, 30, 60, 90, 120, and 180 minutes after glucose treatment. BGLs were measured using Glucocard Test Strip II (Arkray Inc., Kyoto, Japan). Intraperitoneal glucose tolerance test (IPGTT) was performed after the 4-week drug treatment in nondiabetic control and db/db mice. On test days, the animals were fasted for 12 hours and then given an intraperitoneal injection of glucose (500 mg/kg). BGLs were measured in tail blood samples at 0, 15, 30, 60, 90, 120, and 180 minutes after the glucose treatment.
Collection of blood and internal organ samples
At the conclusion of the study, animals were anaesthetized (nembutal, 100 mg/kg), and a 500-µL blood sample was drawn from the orbital venous plexus into a heparinized tube and immediately chilled on ice. The plasma was then separated and stored at -80
before analysis. After blood sampling, visceral adipose tissues (perirenal, retroperitoneal, and epididymal depots), liver, pancreas, and hypothalamus were dissected. The hypothalamus tissues were immediately frozen in liquid nitrogen and stored at -80
until measurement of mRNA levels by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Visceral adipose tissues were weighed and immediately frozen in liquid nitrogen. The liver and pancreas were fixed immediately in 10% neutral formalin solution and then embedded in paraffin.
Serum samples were obtained from blood by centrifugation for 15 minutes at 950 g at room temperature. Fasting glucose and insulin were measured using commercial kits (SpinReact, Gerona, Spain; Millipore, Billerica, MA, USA, respectively). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the final blood glucose and insulin levels in food-deprived mice.
Real-time polymerase chain reaction analysis
Each whole mouse hypothalamus was homogenized in TRI reagent (Sigma-Aldrich) using Ultra-Turrax T25 (IKA, Staufen, Germany). RNA was reverse transcribed to cDNA from 1 µg of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed using 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
for 10 minutes, followed by 45 cycles of 95
for 15 seconds, 55
for 20 seconds, and 72
for 35 seconds. Primers for mouse neuropeptide Y (NPY) and GAPDH were based on NCBI's nucleotide database and designed using the Primer Express program (Applied Biosystems): mouse NPY (forward, 5'-TAT CCC TGC TCG TGT GTT TG-3'; reverse, 5'-GTT CTG GGG GCA TTT TCT G-3'), mouse GAPDH (forward, 5'-CAG GAT GAC ACC AAA ACC CTC-3'; reverse, 5'-TCC AAG CCA GTG ACC CTC TG-3').
Liver and pancreas histopathology
Embedded liver and pancreas tissue blocks were cut into 6-micron sections and stained with hematoxylin and eosin. Pancreas tissue blocks were stained with HRP conjugated anti-insulin antibody. Mounting medium and cover slips were placed on the slides, which were then left to dry overnight. A diagnosis of fatty liver was made based on the presence of macrovesicular or microvesicular fat in 0.5% of the hepatocytes in a given slide.
Tissue lipid determination
Samples of liver were homogenized in 0.25% sucrose containing 1 mM ethylenediaminetetraacetic acid. Lipids were extracted using chloroform/methanol methanol (2:1, v/v) and evaporated in Savant Speedvac Concentrator (Thermo Fisher Scientific Co., Waltham, MA, USA). Next, the pellets were dissolved in 5% fatty acid-free bovine serum albumin dissolved in water. Proteins in the homogenate were assayed using protein assay reagent (Bio-Rad, Hercules, CA, USA) to normalize the amount of lipid extracted. Tissue triglyceride levels were determined using kits from Roche Diagnostics.
The results are expressed as the mean±standard error. SPSS version 14.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. The area under the curve (AUC) was calculated using Microcal Origin software version 7.0 (Microcal Software, Northampton, MA, USA). Comparisons between the two groups were performed with Student two-tailed t-test. When comparing two or more groups, significance was tested using ANOVA with Bonferroni correction to deal with relatively small number of samples. P values of less than 0.05 were considered significant.