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Influence of green tea on enzymes of carbohydrate metabolism, antioxidant defense, and plasma membrane in rat tissues

Nutrition, 2007
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ELSEVIER Nutntion 23 (2007) 687-695 NUTRITION www elsevier coni/locate/nut Basic nutritional investigation Influence of green tea on enzymes of carbohydrate metabolism, antioxidant defense, and plasma membrane in rat tissues Sara Anees Khan, M.Sc, Shubha Priyaravada, M.Sc, Natarajan A. Arivarasu, M.Sc, Sheeba Khan, M.Sc, and Ahad Noor Khan Yusufi, Ph.D.* Department oj BwLhemistr\. hacult\ of Life Sciences Ahgmh Muslim Unwersity Aligarh Ultni Piadesh, India Manuscript received January 11. 2007, accepted June 12, 2007 Abstract Objective: Green tea, consumed worldwide since ancient times, is considered beneficial to human health. We hypothesized that green tea would enhance antioxidant defenses and specific metabolic activities of rat intestine, liver, and kidney to improve their fimctions Methods: The effect of green tea given to rats in the diet or drinking water for 25 d was determined on blood chemistry and on activities of enzymes of carbohydrate metabolism, brush border membrane, and antioxidant defense Results: Senim glucose, cholesterol, phosphate, and body weight decreased, whereas the activities of lactate and malate dehydrogenases and glucose-6- and fructose 1,6-bis-phosphatases increased m the intestine and kidney but slightly changed m the liver Activity of glucosc-6-phosphatc dehy- drogenase profoundly increased m the renal cortex but decreased in other tissues Lipid peroxidation increased in the intestine and renal medulla and decreased in the renal cortex and liver, catalase increased in all tissues but the medulla. Superoxide dismutase activity decreased in the intestine but increased in renal tissues Activities of brush border membrane enzymes in general increased in the intestine and kidney. Conclusion: Green tea consumption resulted m enhanced enzyme activities of carbohydrate metabolism and antioxidant defenses, which may lead to improved health. © 2007 Elsevier Inc All rights reserved. Kewvords Green lea (Camellia sinensis): Carbohydrate metabolism. Antioxidant enzymes. Intestine, Liver, Kidney Introduction Tea in the form of green tea (GT) or black tea is one of the most widely consumed beverages in the world today second only to water [1]. Since ancient times GT consump- tion has been known to maintain and improve health. Poly- This work was supported by a Junior Research Fellowship from the Council of Scientific and Industrial Research, New Delhi, India to Sara Anees Khan, M Sc , and a Junior Research Fellowship/Senior Research Fellowship to Shubha Pnyamvada, M.Sc, from the Indian Council of Medical Research, New Delhi, India. Financial support to the department from the Umversity Grant Commission (UGC-DRF), the Department of Science and Technology (DST-FIST), and research grant 58/2I/2001-BMS from the Indian Council of Medical Research to Ahad Noor Khan Yusufi, Ph.D , IS gratefully acknowledged * Corresponding author Tel +91-571 270-0741, fax -1-91-571-270- 6002 E mail address yusufi@lycos com (A N K Yusufi) phenols are plant metabolites occurring widely in plant food and exhibit outstanding antioxidant and free radical scav- enging properties [2]. GT is an excellent source of polyphe- nols such as catechins [3], orgallotannins, flavonols, flavan- diols, and phenolic acids [4]. In pailicular GT catechins and their derivatives are known to contribute beneficial health effects ascribed to tea by their antioxidant [5], antimuta- genic, and anticarcinogenic properties [6]. GT consumption has been linked to lowering of various forms of cancers [7,8]. GT constituents also have been shown to have car- dioprotective, neuroprotective, antidiabetic, and antimicro- bial properties [9,10]. In addition, GT has been found to be useful in the treatment of arthritis, high cholesterol levels, infection, and impaired immune function [11]. GT con- sumption also has resulted in improved kidney functions in animal models of renal failure [12,13]. We hypothesized that GT enhances specific metabolic activities and anti- 0899-9007/07/$ - see front matter ( dor.lO 1016/j nut 2007 06 007 ' 2007 Elsevier Inc. All rights reserved
688 S. A. Khan et al. / Nutrition 23 (2001) 687-695 oxidant defenses in various rat tissues due to its intrinsic free radical scavenging properties to improve their func- tioning. To address this hypothesis the effect of GT consumption was investigated on the activities of certain enzymes of carbohydrate metabolism, brush border mem- branes (BBMs), lysosomes, and antioxidant defense system in the rat model. Green tea consumption resulted in decreases in serum gluco.se, cholesterol, and inorganic phosphate (Pi) and an increase in semm phospholipids. The activities of enzymes of glucose oxidation and its production selectively but vari- ably increased in intestine and kidney tissues. Lipid peroxi- dation (LPO), an indicator of cell injury, and enzyme ac- tivities of the antioxidant defense system, e.g., superoxide dismutase (SOD) and catalase, and BBM were also altered but differentially in different tissues by GT consumption. The results indicate an overall improvement in metabolic activities in various tissues most likely by GT-polyphenol- mediated reduction of oxidative damage under normal phys- iologic conditions in the rat model. Materials and methods Materials Green tea (Kangra, Himanchal Pradesh, India and Lip- ton-Unilever, Englewood Cliffs, NJ, USA) was purchased from commercial sources (Jain Pan House, New Delhi, India). All other chemicals used were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA) or Sisco Research Laboratory (Mumbai, India). Experimental design The animal experiments were conducted according to the guidelines of the Committee for Purpose of Control and Supervision of Experiments on Animals, Ministry of Envi- ronment and Forests, Government of India. Adult male Wistar rats, weighing 150-180 g, fed with a standard pellet diet (Aashirwad Industries, Chandigarh, India) and water ad libitum, were conditioned for 1 wk before the start of the experiment. All animals were kept under conditions that prevented them from experiencing unnecessary pain and discomfort according to guidelines approved by the ethical committee. A known amount of diet (250 g) and waler/GT extract (2 X 125 mL = 250 mL/d) was provided in two servings to each group of rats and was found to be sufficient. Three groups of rats (six to eight rats/group/cage) were studied. The rats of group 1 (control) received the standard diet and water for 25 d. The rats of group 2 (GT extract) were given the standard diet and GT extract (3% w/v) in drinking water, whereas the rats of group 3 (GT diet) re- ceived GT in the diet (3% w/w) and 250 mL of water as mentioned above also for 25 d. After 25 d of tea adminis- tration, the rats were sacrificed under light ether anesthesia. Blood samples were collected from non-fasted rats and the liver, kidney, and entire intestine (starting from the ligament of Trietz to the end of ileum) were extracted. The intestines were washed by flushing them with ice-cold buffered saline (1 niM Tris-HCl, 9 g/L of NaCI, pH 7.4). The liver and kidney were placed in Tris buffered saline. All preparations and analyses of various parameters were carried out simul- taneously under similar experimental conditions to avoid any day-to-day variations. Preparation of GT extract and GT diet Green tea (30 g ) was added to 500 mL of boiling water and was steeped for 15-20 min. Infusion was cooled to room temperature and then filtered. The tea leaves were extracted a second time with 500 mL of boiling water and filtered, and the two filtrates were combined to obtain 3% GT extract (3 g of tea leaves/100 mL of H^O). The resulting clear solution is similar to tea brews consumed by humans. The GT diet was prepared by adding GT (3 g/100 g of diet) in powdered rat diet, mixed well with small amounts of water, and then made in the form of cookies, after drying in an oven (50-60°C) for 30 min. GT polyphenols were de- tected by thin layer chromatography as reported by Malik et al. [14]. According to the manufacturer's information, the antioxidant content was 95 mg/g of GT. Preparation of homogenates The washed intestines were slit in the middle and the entire mucosa was gently scraped with a glass slide and weighed. A 6.5% homogenate of this mucosa was prepared in 50 mM mannitol, pH 7.0, in a glass Teflon homogenizer (Remi Motors, Mumbai, India) with five complete strokes. The homogenate was then subjected to a high-speed Ultra- Turrex homogenizer (Type T-25, Janke & Kunkel GMBH & Co. KG., Staufen, Germany) for three pulses of 30 s each with an interval of 30 s between each stroke. The kidneys were decapsulated and kept in ice-cold 154 mM NaCl and 5 mM Tris-HEPES buffer, pH 7,5. The cortical and medullary regions were carefully separated and homogenized (as mentioned above) in 50 mM mannitol buffer to obtain 10% (w/v) homogenate. The 10% liver homogenate was similarly prepared in 10 mM Tris-HCl buffer, pH 7.5. One part of the homogenates (of intestine, kidney, and liver) was centrifuged at 2000g for 10 min at 4°C and the supernatant was saved for assaying the enzymes of car- bohydrate metabolism; the second part was centrifuged at 3000g for 15 min at 4°C and the supernatant was used for assay of free radical scavenging enzymes; and the third part was used for estimation of total thiol (-SH) and LPO.
NUTRITION ELSEVIER Nutntion 23 (2007) 687-695 www elsevier coni/locate/nut Basic nutritional investigation Influence of green tea on enzymes of carbohydrate metabolism, antioxidant defense, and plasma membrane in rat tissues Sara Anees Khan, M.Sc, Shubha Priyaravada, M.Sc, Natarajan A. Arivarasu, M.Sc, Sheeba Khan, M.Sc, and Ahad Noor Khan Yusufi, Ph.D.* Department oj BwLhemistr\. hacult\ of Life Sciences Ahgmh Muslim Unwersity Aligarh Ultni Piadesh, India Manuscript received January 11. 2007, accepted June 12, 2007 Abstract Objective: Green tea, consumed worldwide since ancient times, is considered beneficial to human health. We hypothesized that green tea would enhance antioxidant defenses and specific metabolic activities of rat intestine, liver, and kidney to improve their fimctions Methods: The effect of green tea given to rats in the diet or drinking water for 25 d was determined on blood chemistry and on activities of enzymes of carbohydrate metabolism, brush border membrane, and antioxidant defense Results: Senim glucose, cholesterol, phosphate, and body weight decreased, whereas the activities of lactate and malate dehydrogenases and glucose-6- and fructose 1,6-bis-phosphatases increased m the intestine and kidney but slightly changed m the liver Activity of glucosc-6-phosphatc dehydrogenase profoundly increased m the renal cortex but decreased in other tissues Lipid peroxidation increased in the intestine and renal medulla and decreased in the renal cortex and liver, catalase increased in all tissues but the medulla. Superoxide dismutase activity decreased in the intestine but increased in renal tissues Activities of brush border membrane enzymes in general increased in the intestine and kidney. Conclusion: Green tea consumption resulted m enhanced enzyme activities of carbohydrate metabolism and antioxidant defenses, which may lead to improved health. © 2007 Elsevier Inc All rights reserved. Kewvords Green lea (Camellia sinensis): Carbohydrate metabolism. Antioxidant enzymes. Intestine, Liver, Kidney Introduction Tea in the form of green tea (GT) or black tea is one of the most widely consumed beverages in the world today second only to water [1]. Since ancient times GT consumption has been known to maintain and improve health. PolyThis work was supported by a Junior Research Fellowship from the Council of Scientific and Industrial Research, New Delhi, India to Sara Anees Khan, M Sc , and a Junior Research Fellowship/Senior Research Fellowship to Shubha Pnyamvada, M.Sc, from the Indian Council of Medical Research, New Delhi, India. Financial support to the department from the Umversity Grant Commission (UGC-DRF), the Department of Science and Technology (DST-FIST), and research grant 58/2I/2001-BMS from the Indian Council of Medical Research to Ahad Noor Khan Yusufi, Ph.D , IS gratefully acknowledged * Corresponding author Tel +91-571 270-0741, fax -1-91-571-2706002 E mail address yusufi@lycos com (A N K Yusufi) 0899-9007/07/$ - see front matter ( ' 2007 Elsevier Inc. All rights reserved dor.lO 1016/j nut 2007 06 007 phenols are plant metabolites occurring widely in plant food and exhibit outstanding antioxidant and free radical scavenging properties [2]. GT is an excellent source of polyphenols such as catechins [3], orgallotannins, flavonols, flavandiols, and phenolic acids [4]. In pailicular GT catechins and their derivatives are known to contribute beneficial health effects ascribed to tea by their antioxidant [5], antimutagenic, and anticarcinogenic properties [6]. GT consumption has been linked to lowering of various forms of cancers [7,8]. GT constituents also have been shown to have cardioprotective, neuroprotective, antidiabetic, and antimicrobial properties [9,10]. In addition, GT has been found to be useful in the treatment of arthritis, high cholesterol levels, infection, and impaired immune function [11]. GT consumption also has resulted in improved kidney functions in animal models of renal failure [12,13]. We hypothesized that GT enhances specific metabolic activities and anti- 688 S. A. Khan et al. / Nutrition 23 (2001) 687-695 oxidant defenses in various rat tissues due to its intrinsic free radical scavenging properties to improve their functioning. To address this hypothesis the effect of GT consumption was investigated on the activities of certain enzymes of carbohydrate metabolism, brush border membranes (BBMs), lysosomes, and antioxidant defense system in the rat model. Green tea consumption resulted in decreases in serum gluco.se, cholesterol, and inorganic phosphate (Pi) and an increase in semm phospholipids. The activities of enzymes of glucose oxidation and its production selectively but variably increased in intestine and kidney tissues. Lipid peroxidation (LPO), an indicator of cell injury, and enzyme activities of the antioxidant defense system, e.g., superoxide dismutase (SOD) and catalase, and BBM were also altered but differentially in different tissues by GT consumption. The results indicate an overall improvement in metabolic activities in various tissues most likely by GT-polyphenolmediated reduction of oxidative damage under normal physiologic conditions in the rat model. Materials and methods Materials Green tea (Kangra, Himanchal Pradesh, India and Lipton-Unilever, Englewood Cliffs, NJ, USA) was purchased from commercial sources (Jain Pan House, New Delhi, India). All other chemicals used were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA) or Sisco Research Laboratory (Mumbai, India). Experimental design The animal experiments were conducted according to the guidelines of the Committee for Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India. Adult male Wistar rats, weighing 150-180 g, fed with a standard pellet diet (Aashirwad Industries, Chandigarh, India) and water ad libitum, were conditioned for 1 wk before the start of the experiment. All animals were kept under conditions that prevented them from experiencing unnecessary pain and discomfort according to guidelines approved by the ethical committee. A known amount of diet (250 g) and waler/GT extract (2 X 125 mL = 250 mL/d) was provided in two servings to each group of rats and was found to be sufficient. Three groups of rats (six to eight rats/group/cage) were studied. The rats of group 1 (control) received the standard diet and water for 25 d. The rats of group 2 (GT extract) were given the standard diet and GT extract (3% w/v) in drinking water, whereas the rats of group 3 (GT diet) received GT in the diet (3% w/w) and 250 mL of water as mentioned above also for 25 d. After 25 d of tea administration, the rats were sacrificed under light ether anesthesia. Blood samples were collected from non-fasted rats and the liver, kidney, and entire intestine (starting from the ligament of Trietz to the end of ileum) were extracted. The intestines were washed by flushing them with ice-cold buffered saline (1 niM Tris-HCl, 9 g/L of NaCI, pH 7.4). The liver and kidney were placed in Tris buffered saline. All preparations and analyses of various parameters were carried out simultaneously under similar experimental conditions to avoid any day-to-day variations. Preparation of GT extract and GT diet Green tea (30 g ) was added to 500 mL of boiling water and was steeped for 15-20 min. Infusion was cooled to room temperature and then filtered. The tea leaves were extracted a second time with 500 mL of boiling water and filtered, and the two filtrates were combined to obtain 3% GT extract (3 g of tea leaves/100 mL of H^O). The resulting clear solution is similar to tea brews consumed by humans. The GT diet was prepared by adding GT (3 g/100 g of diet) in powdered rat diet, mixed well with small amounts of water, and then made in the form of cookies, after drying in an oven (50-60°C) for 30 min. GT polyphenols were detected by thin layer chromatography as reported by Malik et al. [14]. According to the manufacturer's information, the antioxidant content was 95 mg/g of GT. Preparation of homogenates The washed intestines were slit in the middle and the entire mucosa was gently scraped with a glass slide and weighed. A 6.5% homogenate of this mucosa was prepared in 50 mM mannitol, pH 7.0, in a glass Teflon homogenizer (Remi Motors, Mumbai, India) with five complete strokes. The homogenate was then subjected to a high-speed UltraTurrex homogenizer (Type T-25, Janke & Kunkel GMBH & Co. KG., Staufen, Germany) for three pulses of 30 s each with an interval of 30 s between each stroke. The kidneys were decapsulated and kept in ice-cold 154 mM NaCl and 5 mM Tris-HEPES buffer, pH 7,5. The cortical and medullary regions were carefully separated and homogenized (as mentioned above) in 50 mM mannitol buffer to obtain 10% (w/v) homogenate. The 10% liver homogenate was similarly prepared in 10 mM Tris-HCl buffer, pH 7.5. One part of the homogenates (of intestine, kidney, and liver) was centrifuged at 2000g for 10 min at 4°C and the supernatant was saved for assaying the enzymes of carbohydrate metabolism; the second part was centrifuged at 3000g for 15 min at 4°C and the supernatant was used for assay of free radical scavenging enzymes; and the third part was used for estimation of total thiol (-SH) and LPO. 689 S A Khan el al /Nutrition 23 (2007) 687-695 Preparation of BBM Intestinal BBM was prepared at 4°C using dvfferential precipitation by CaC^ [15]. Mucosa scraped from four to five washed intestines was used for each BBM preparation. CaClj was added to the homogenate to a final concentration of 10 mM and the mixture was stirred for 20 min on ice. The final membrane preparations were suspended in 50 mM sodium maleate buffer, pH 6.8. Kidney BBM was prepared from whole cortex homogenate using the MgClj precipitation method as described by Yusufi and Dousa [16]. The final membrane preparations were suspended in 300 mM mannitol, pH 7.4, and the BBMs thus prepared were saved and stored at -20°C until further analysis for BBM enzymes. Each sample of BBM was prepared by pooling tissues from two to three rats. Serum chemistries Serum samples were deproteinated with 3% trichloroacetic acid at a ratio of 1.3, left for 10 min, and then centrifuged at 2000g for 10 min. The protein-free supernatant was used to determine Pi. The precipitate was used to quantitate total phospholipids. Serum urea nitrogen and cholesterol levels were determined directly in serum samples. Glucose was estimated by an o-toluidine method using a kit from Span Diagnostics (Mumbai, India). These parameters were determined by standard procedures as mentioned in a previous study [17]. Assay of carbohydrate metabolism enzymes The activities of the enzymes involving oxidation of reduced nicotinamide adenine dinucleotide or reduction of nicotinamide adenine dinucleotide phosphate were determined spectrophotometrically on a Cintra 5 (GBC Scientific Equipment, Pty, Victoria, Australia) fixed for 340 nm using 3 mL of assay in a 1-cm cuvette at room temperature (28-30°C). The enzyme assays of lactate dehydrogenase (LDH; E.C. 1.1.1.27), malate dehydrogenase {E.C. 1.1.1.37), malic enzyme (ME; E.C. 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH; E.G. 1.1.1.49), glucose-6-phosphatase (G6Pase; EC.3.1.3.3), and fmctose-l, 6-bisphosphatase (FBPase; E.C.3.1.3.11) activities were studied as described by Khundmiri et al. [18]. Hexokinase was estimated by the method of Crane and Sols [19] and the remaining glucose was measured by method of Nelson [20]. Assay of BBM marker enzymes and lysosomal marker enzymes The activities of alkaline phosphatase (ALP), leucine amino peptidase (LAP), y-glutamyl transferase (GGTase), sucrase, and acid phosphatase (ACPase) were determined as described by Farooq et al. [21]. Assay of enzymes involved in free radical scavenging Superoxide dismutase (B.C. 1.15.1.1) was assayed by the method of Marklund and Marklund [22]. Catalase (B.C. 1.11.1. 6) activity was assayed by the method of Giri et al. [23]. LPO and total -SH group estimation Total SH groups were determined by the method of Sedlak and Lindsay [24] and LPO by the method of Ohkawa et al. [25]. Definition of unit One unit of enzyme activity is the amount of enzyme required for the formation of 1 fi-mol of product per hour under specified experimental conditions. Specific activity is enzyme units per milligram of protein. Statistical analysis All results are expressed as mean ± SEM for at least three to four separate preparations. The data were analyzed for statistical significance by Student's t test for group comparisons or by analysis of variance. P < 0.05 was considered statistically significant. Results Considering reported numerous health benefits of GT, the effect of GT given in the diet (GT diet) or as tea extract (GT extract) in drinking water was determined on certain enzymes involved in various pathways of carbohydrate metabolism, biomarkers of renal and intestinal BBMs, and various parameters of antioxidant defense mechanism were determined in different rat tissues used as a model for humans. Effect of GT on body weight and serum parameters In general, the rats remained active and alert throughout the study. The daily food and fluid intakes were similar in Table 1 Effect of GT consumption via drinking fluid or diet on body weight (grams) of rats* Groups Before treatment After treatment % Change Control GT extract GT diet 1667 + 5 3 168 8 ± 6 3 164 3 ± 7 4 162 5 ± 8 53 150 ± 7 9 145 ± 10* -2 5 -11 I -11 7 GT, green tea * Results are expressed as mean ± SEM * Significantly different at P < 0 05 from control S A Khan el al I Numtion 23 (2007) 687-695 690 Table 2 Effects of GT consumption via drinking fluid or diet on serum parameters* Groups BUN (mg/dU Glucose (mg/dL) Cholesterol (mg/dL) Inorganic PO^ (/xmol/mL) Phospholipid (/j,gs/mL) Control GT extract GTdiet 25 43 ± 0 36 20 99 ± 0 61* (-17%) 2 1 0 9 + 1 17* (-17%) 141 09 ± 4 64 116 11 ± 5 62* (-18%) 107 78 ± 6 89* (-24%) 137 60 + 30 20 103 97 ± 2 03 (-24%) 98 35 ± 2 80 (-29%) 164 ± 0 04 1 29 + 0 04* (-21%) 1 07 + 0 14* (-35%) 109 4 0 = I 96 162 0 0 = 13 8" (+48%) 14190 = 9 30' (+30%) BUN, serum urea nitrogen, GT, green tea * Results are mean + SEM of six different samples Values in parentheses represent percent change from control * Significantly different at P < 0 05 from control control and GT rats (data not shown). The amount of GT ingested m the diet or by drinking was approximately the same. As shown in Table 1, GT consumption resulted in slight loss of body weight (—11%) compared with control rats. Serum glucose, cholesterol, and Pi significantly declined, whereas phospholipids significantly increased by both forms of GT consumption. Serum urea nitrogen was slightly lowered (Table 2). The changes caused by the GT diet or GT extract on various parameters varied in magnitude, but the changes were always in the same direction. renal cortical homogenates. The activities of ALP, GGTase, and ACPase also slightly increased, whereas LAP decreased in the renal medulla. In liver homogenates ALP and GGTase decreased, whereas LAP and ACPase were not affected. Similar to homogenates, the activity of ALP decreased, whereas GGTase, LAP, and sucrase activities significantly increased in intestinal BBM by GT consumption (Table 4). The activities of ALP and GGTase increased, whereas LAP activity significantly lowered in BBM vesicles isolated from the renal cortex (Table 4) Effect of GT on biomarkers of BBM and lysosomes in different tissues Effect of GT on enzymes of carbohydrate metabolism The influence of GT consumption was determined on BBM and lysosomal enzymes in the liver, intestine, and renal cortical and medullary homogenates and in BBM vesicles isolated from the renal cortex and intestinal mucosa. The results summarized in Table 3 show that the activities of ALP, GGTase, LAP, sucrase, and ACPase significantly increased in intestinal homogenates m GT compared with control rats. The activities of ALP and ACPase increased, whereas GGTase and LAP decreased in The effect of GT consumption was determined on the activities of carbohydrate metabolism enzymes in the intestine, liver, and kidney. The activity of hexokinase slightly increased in the liver (-^25%) and renal medulla (+20%) when GT was given in the diet and in the intestine (+18%) when given in the drinking water but did not change in the renal cortex with either method. However, the activity of LDH markedly increased m the intestine and kidney tissues but only slightly increased m the liver. The effect on LDH activity was more expressive in GT extract than in GT diet Table 3 Effects of GT consumption via dnnking fluid or diet on brush border membrane enzymes in different tissue homogenates* Tissue Intestine Control GT extract GT diet Liver Control GT extract GT diet Cortex Control GT extract GT diet Medulla Control GT extract GT diet ALP (/xmol/mg protein/h) GGTase (/i.mol/mg protein/h) LAP (;j.mol/mg protein/h) ACPase (^imol/mg protein/h) Sucrase (/xmol/mg protein/h) 7 26 ± 0 15 3 50 ± 0 08 8 45 ± 0 3 93±0 22 53 ± 0 88 24 46 ± 1 45 (+9%) 25 99 ± 1 36 (+15%) 1 30 ± 0 04 161 ± 0 0 2 t ( + 2 4 % ) 1 38 ± 0 06 (+6%) 4 46 ± 0 14 6 63 ± 0 26t (+49%) 4 9 4 ± 0 15* (+11%) 9 07 ± 0 09* (+25%) 4 97 ± 0 15* (+42%) 124 ± 0 0 3 103 ± 0 0 6 * ( - 1 7 % ) 105 ± 0 06* (-15%) 2 25 ± 0 0 8 1 12 ± 0 13* (-50%) 1 48 ± 0 08* (-34%) 0 59 ± 0 02 063 ± 0 02 (+7%) 0 59 ± 0 03 7 33 ± O 24 7 07 ± 0 35 (-4%) 6 89 ± 0 50 ( - 6 0%) 2 93 ± 0 14 3 5 6 ± 0 11* (+22%) 3 35 ± 0 08* (+14%) 40 52 ± 0 56 37 42 ± 0 65* (-8%) 33 54 ± 129* (-17%) 4 75 ± 0 32 3 18 ± 0 27* (-33%) 2 91 ± 0 19" (-39%) 604 ± 0 14 6 40 ± 0 22 (+6%) 7 93 ± 0 33" (+31%) 1 71 ± 0 1 2 07 ± 0 18* (+21%) 171 ± 0 08 10 59 ± 0 41 1264 ± 0 7 2 * ( + 19%) 10 65 ± 0 54 (+0 6%) 1304±081 7 19 ± 0 22* (-45%) 7 44 ± 0 22* (-43%) 6 29 ± 0 26 7 53 ± 0 2'(+20%) 8 06 ± 0 39* (+28%) 14T(+16%) 15T(+12%) ACPase, acid phosphatase, ALP, alkaline phosphatase, GGTase, y-glutamyl transferase, GT, green tea, LAP, leucine amino peptidase * Results are mean ± SEM of three to four different preparations Values in parentheses represent percent change from control * Significantly different at P < 0 05 from control 5 A Khan el al /Nutrition 23 (2007) 687-695 I compared with control rats in the intestine (+672%) and renal cortex (+111%) The activity of maldte dehydrogenase, an en/yme of the Tricarboxylic acid cycle (TCA) cycle, was similarly enhanced in the intestine ( + 31%) and renal cortex (+67%) to a greater extent by GT extiact than by GT diet but was slightly lowered in the renal medulla (-18%; Table 5). The effect of GT was also determined on the activities of enzymes involved in gluconeogenesis and the hexose monophosphate (HMP)-shunt pathway (Table 6) The activities of G6Pase, FBPase, G6PDH, and ME changed similarly with the GT extract and the GT diet in all tissues. The activity of G6Pase significantly increased in the intestine (+34% to +42%), renal cortex (+13% to +16%), and medulla (+27% to +43%) but decreased in the liver (-13% to -20%). The activity of FBPase also increased but to a much lesser extent than G6Pase in these tissues. The activity of FBPase similarly to G6Pase was lowered in the hver. The activity of G6PDH (HMP-shunt) significantly decreased in the intestine (-40% to -48%), liver ( - 7 3 % to -79%), and renal medulla (-32% to -58%) but profoundly increased in the renal cortex (+121% to +146%) The activity of ME, which is a source of cellular reduced nicotinamide adenine dinucleotide phosphate, together with G6PDH significantly decreased in all tissues except the renal cortex. It should be noted that the activities of various metabolic enzymes studied in the liver altered in a narrow range except G6PDH, which was significantly lowered by GT consumption. I -^ r-i o +1 +1 +1 vo r^ «n — r-- 00 00 PO O Tf CM (N oo rs a\ m t^ " fS \£) (N (N (N +1 +1 +1 s o t ^ ON r-- — cs O e^ O r^ ro ^ -rr in -^ r f \D Tt (N m (N o o o +1 +1 +1 oo r- — ON CL, < O ++ 00 * n — CM +1 +1 +1 ON 0 11 II ^ t^ 691 <D Ejfect of GT on activities of antioxidant enzymes and related parameters of oxidative stress —o 0 0 ON + + PQ PO DC -^ e OO m Ov r n (N — J3 U -as +i +1 +1 (N 0\ o o r^ 55 :^ > •«* O v Ov 03 ++ DO 8l o o — +1 +1 +1 g o- n o S _ o - t ; i3 •2 w- w0 u -5 « 3 ^ -P G Ou — so Tl O ^ CN ^ V-) — o o o +1 +i +1 £ o tu V 5 ° a. S +1 K Because GT has been reported to exert its biological effects primarily by perturbation of the antioxidant defense system, the activities of SOD and catalase and related parameters of oxidative stress were determined in control and GT rats. It was observed that GT given in the diet or in drinking water caused similar alterations in these parameters. LPO measured as the level of malondialdehyde was significantly lowered in the liver and renal cortex but significantly enhanced in the intestine and renal medulla by GT consumption. Total SH levels were significantly decreased in the liver and renal cortex but significantly increased in the renal medulla in GT compared with control rats. The activity of SOD significantly decreased in the intestine and liver but significantly increased in the renal cortex and medulla (Table 7). In contrast, catalase activity markedly increased in the intestine and liver but did not change in renal tissues by GT ingestion. CL < o n ^1^ 7:; i= a S U M 03 OS (;5 O H BJ CO * CJ O O Discussion -K Tea is the most consumed beverage in the world, aside from water. Studies carried out in laboratories and on S. A. Khan el al. / Nutrition 23 (2007) 687-695 692 Table 5 Effects of GT consumption via drinking fluid or diet on the activity of metabolic enzymes in different tissue homogenates Tissue Intestine Control GT extract GT diet Liver Control GT extract GT diet Cortex Control GT extract GT diet Medulla Control GT extract GT diet Hexokinase (\x.mo\lm% protein/h) LDH (pimol/mg protein/h) 1.13 ±0.27 8.72 ± 0.26* (+672%) 4.98 ± 0.34* ( + 341%) 116.92 ±3.58 138.02 ±6.36^ (+18%) 117.68 ± 5.25 ( + 0.65%) 35.85 ± 2.41 40.41 ±0.99 (+13%) 43.13 ± 1.76* (+20%) 30.08 ± 1.03 30.71 ± 1.13 ( + 2%) 37.68 ± 1.72* (+25%) 6.46 ± 0.56 13.6 ± 1.22* (+111%) 7.23 ±0.46 (+12%) 86.88 ± 2.09 82.39 ± 2.34 (-5%) 80.92 ± 1.46* (-7%) 11.69 ±0.60 15.89 ±0.86* (+36%) 15,83 ± 0.96* (+35%) 66.60 ±1.191 70.13 ± 1.80 (+5%) 80.18 ± 1.87* (+20%) MDH (/umol/mg protein/h) 21.52 ± 1.29 28.11 ±4.24 ( + 31%) 22.41 ± 1.38 (^ 4%) 3.78 ± 0.24 3.97 ± 0.38 ( + 5%) 4.58 ±0.14* ( + 21%) 23.26 ± 0.90 38.92 ± 5.35* (-f 67%) 33.43 ± 2.66* (+44%) 26.99 ± 0.65 22.24 ± 1.85* (-18%) 26.17 ± 1.25 (-3%) GT, green tea; LDH, lactate dehydrogenase; MDH, malate dehydrogenase * Results are mean ± SEM of three to four different preparations. Values in parentheses represent percent change from control. * Significandy different at P < 0.05 from control. animaKs have suggested thai GT in particular has extensive health benefits. Most of these effects are considered to be due to the presence of chiefly tea catechins, polyphenolic compounds [8] that exhibit antioxidative, antiinflammatory, anticarcinogenic, anti-arteriosclerotic, and antibacterial effects [9]. We propose that GT polyphenols and other constituents cause specific adaptive alterations in the cellular metabolism of certain tissues and thus improve their functioning. To address this proposal, the rats were given GT in the diet or in drinking water and the activities of various enzymes involved in carbohydrate metabolism, antioxidant defense system, and BBM were determined in the intestine, liver, and renal tissues. In general, GT consumption resulted in slight loss of body weight that was associated with lowering of blood glucose, cholesterol, and Pi. These observations are in partial agreement with some previous reports in humans and animals [26]. Oral administration of GT has been shown to decrease plasma total cholesterol and blood triacylglycerols and serum glucose in fasted and nonfasted human subjects [27,28]. The suppressive effects of GT catechins on the postprandial levels of triacylglycerols and cholesterol have also been reported in humans [29]. Taken together, these observations may be respon- Table 6 Effects of GT consumption via drinking fluid or diet on the activity of metabolic enzymes in different tissue homogenates* Tissue Intestine Control GT extract GT diet Liver Control GT extxact. GT diet Cortex Control GT extract GT diet Medulla Control GT extract GT diet G6Pase (/xmol/mg protein/h) FBPase (/bimol/mg protein/h) G6PDH (/xmol/mg protein/h) ME ((xmol/mg protein/h) 1.96 ± 0.05 2.79 ±0.17* (+42%) 2.62 ±0.13* (+34%) 2.70 ± 0.09 2.89 ±0.09 (+7%) 2.81 ±0.06 (+4%) 1.08 ±0.05 0,56 ±0.13* (-48%) 0.65 ± 0.08* (-40%) 0.68 i 0.09 0.35 - 0.06* (-49%) 0.42 ± 0.05* (-38%) 0.15 ± 0.002 0.12 ±0.002* (-20%) 0.13 ±0.005* (-13%) 0.85 ± 0.02 0.77 ± 0.02* (-9%) 0.75 ±0.03* (-12%) 3.23 ±0.21 0.87 ±0.11* (-73%) 0.69 ±0 . 1 1 ' ( - 7 9 % ) 0.69 ± 0.05 0.60 ± 0.06 (-13%) 0.58 ±0.07 (-16%) 0.56 ± 0.01 0.65 ±0,02* (+16%) 0.63 ±0.01* (+13%) 2.57 ± 0.05 2.85 ±0.06* ( + 11%) 2.72 ±0.15 (+6%) 0.24 ± 0.02 0.53 ±0.03* (+121%) 0.59 ±0.01* (+146%) 0.45 ± 0.08 0.44 r 0.06 (-2%) 0.42 ± 0.09 (-7%) 0.45 ± 0,01 0.57 ± 0,02* ( + 27%) 0.64 ±0,02* (+43%) 1.76 ±0.04 2.08 ±0.12* (+18%) 2.05 ±0.06* ( + 17%) 0.57 ± 0.05 0.24 ± 0.04* (-58%) 0.39 ±0.01 (-32%) 1.53 ± 0.11 1.19 ± a i 5 ( - 2 2 % ) 0.86 = 0.09* (-44%) FBPase, fructose-1,, 6-bisphosphatase; G6Pase, glucose-6-phosphatase; G6PDH, glucose -6-phosphate dehydrogenase; GT, green tea; ME. malic enzyme * Results are mean ± SEM of three to four different preparations. Values in parentheses represent percent change from control. * Significantly different at P < 0.05 from control. 693 S A Khan ei al / Numiion 23 (2007) 687-695 Table? Effects of GT consumption via dnnking fluid or diet on enzymic and non enzymic antioxidant parameteri, in different tissue homogenates* Tissue Intestine Control GT extract GT diet Liver Control GT extract GT diet Cortex Control GT extract GT diet Medulla Control GT extract GT diet LPO (nmol/gm tissue) Total - S H (/imol/gm tissue) SOD (Units/mg protein) Catalase (jxmol/ing protein/mm) 6142 ± 2 60 87 20 ± 6 75" (+42%) 105 60 ± 7 04* (+72%) 1 88 ± 0 05 2 00 ± 0 08 (+6%) 2 45 ± 0 08* (+30%) 7 04 ± 0 63 4 73 ± 0 40" (-33%) 3 40 ± 0 30* (-52%) 2 90 ± 0 55 6 30 ± 0 45 (+117%) 10 79 ± I 23* ( + 272%) 408 20 ± 43 20 265 4 0 + 14 4 0 ' ( - 3 5 % ) 21601 ± 13 80* (-47%) 10 67 ± 0 40 7 60 ± 0 25* (-29%) 6 89 ± 0 59" (-35%) 82 90 ± 1 94 72 9 0 ± 140* (-12%) 75 80 ± I 30" (-9%) 1342 ± 2 15 36 75 ± 5 90 (+174%) 42 88 ± 2 37 ( + 220%) 272 60 ± 20 43 228 00 ± 16 00 (-16%) 171 70 ± 21 90" (-37%) 7 49 ± 0 38 5 73 ± 0 22* (-24%) 6 42 ± 0 75 (-14%) 12 31 ± 1 24 18 20 ± 1 60* (+48%) 21 31 ± 1 50 (+73%) 108 40 ± 9 80 129 90 ± 5 20 (+20%) 135 9 ± 6 90 ( + 25%) 73 26 ± 5 58 131 70 ± 10 55* (+80%) 89 31 ± 2 00* (+22%) 2 90 ± 0 04 3 64 ± 0 01" (+26%) 4 66 ± 0 2 3 (+61%) 22 92 ± 0 65 28 6 ± 1 82* (+25%) 36 90 ± 0 75* (+61%) 139 40 ± 7 34 1045 ± 3 88 (-25%) 132 03 ± 5 42* (-5%) GT green tea LPO, lipid peroxidation, - S H thiol, SOD, superoxide dismutase * Results are mean ± SEM of three to four different preparations Values in parentheses represent percent change from control * Significantly different at f < 0 05 from control sible for body weight loss and in lowering the incidence of cardiovascular diseases [29,30] The present results further demonstrate that GT causes selective adaptive alterations in the activities of certain enzymes involved in glycolysis, TCA cycle, gluconeogenesis, and HMP-shunt pathway in the intestine, liver, and renal cortex and medulla The activities of hexokinase except m the renal cortex and that of LDH variably increased The acUviUes of malale dehydrogenase, a TCA cycle enzyme, except in the renal medulla, were also increased in the same manner These observations suggest that GT consumption enhanced glucose degradation in the intestine, liver, and kidney albeit to different extents m these tissues The profound increase of LDH activity in the intestine and renal medulla are suggestive of an increase in anaerobic glycolysis as a major source of energy because these tissues function in a low oxygen environment [17] Further, GT consumption resulted in an increase in the activities of G6Pase and FBPase in the intestine and renal tissues, indicating that the production of glucose by gluconeogenesis was also enhanced in these tissues However, the activities of these enzymes significantly decreased in the liver Thus the effect of GT on glucose degradation and its production appear to be tissue specific The effect of GT on carbohydrate metabolism has not been studied in detail in major tissues except m the liver and adipocytes GT consumption increased glucose metabolism in adipocytes [30,31], whereas hepatic glucose production was reported to be inhibited by GT, leading lo lower blood glucose levels [32] A GT polyphenol, epigallocatechin-3gallate, was reported to be an insulin mimetic in that it lowered blood glucose in obese Zucker rats [33,34] Moreover, Waltner-Law et al [32] reported a significant decrease in the expression of genes that control gluconeogenesis such as poshhoenol pyruvate carboxykmase and G6Pase genes m liver cells The observed decrease m the activities of glu coneogenic enzymes (G6Pase and FBPase) in liver homogenates IS compatible with these observations In contrast to the enzymes of glycolysis, TCA cycle, and gluconeogenesis, the activity of G6PDH (HMP shunt) and ME were differentially altered by GT The activity of G6PDH significantly declined in the intestine, liver, and renal medulla but profoundly increased in ihe renal cortex The activity of ME was selectively decreased m the intes tine and renal medulla but not affected in the liver and renal cortex These enzymes act to produce cellular reduced nic otinamide adenine dmocleotide phosphates that play an im portant role in reducing anabolic pathways and the antiox idant defense mechanism The increased activity of G6PDH in the renal cortex might support reducing anabolic reac tions, e g , lipid biosynthesis, and improve the antioxidant defense mechanism to lower oxidative damage In contrast, lower activities of G6PDH and ME in the intestine, liver, and renal medulla may have resulted in lower cholesterol synthesis and thus lower blood cholesterol GT has been shown to increase energy expenditure and fat oxidation in humans [35,36] The observed increase in oxidative metabolism (mitochondrial enzymes) and increased glucose pro duction by gluconeogenesis is in partial agreement with these reports Pi is an essential component of intermediary metabolism, energy conservation as adenosine triphosphate, and biosynthesis of cellular membranes (as phospholipids) and other important biomolecules such as nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and vanous RNAs The decrease m serum Pi may suggest Its overutilization in these processes Moreover, Pi may have been converted to phospholipids as indicated by higher serum phospholipids levels needed for much required synthesis of membranes such as the endoplasmic 694 S. A. Khan et al. /Nutrition 23 (2007) 687-695 reticulum, mitochondria, and plasma membrane to support higher metabolic activities. Most beneficial health effects ascribed to GT are considered to be mediated by potential antioxidant properties of its constituents that scavenge free radicals and reduce oxidative damage [37]. Several lines of evidence suggest that prooxidant and antioxidant actions of plant polyphenols may be important mechanisms for their anticancer properties [38]. Reactive oxygen species (ROS) are normal byproducts of aerobic metabolism. Most intracellular ROS are generated via mitochondrial electron transport, although other normal biological processes contribute. It has been reported that GT exerts its biological effects on the basis of the redox state of a particular cell/tissue [39,40] and according to the level of GT polyphenols accumulated in the tissues [41]. To maintain proper redox balance, many defense systems have evolved. A major cellular defense against ROS is provided by SOD and catalase, which together convert superoxide radicals first to HjOj and then to water and molecular oxygen. Other enzymes, e.g., glutathione (GSH) peroxidase, use the thiol reducing power of glutathione to reduce oxidized lipids and protein targets of ROS. It has been reported that GT causes increases in the activities of catalase, SOD, GSH-peroxidase, and GSH-transferase and reduces LPO in the liver and kidney [42]. The present results demonstrated that GT consumption increased LPO in the intestine and renal medulla but markedly decreased in the liver and renal cortex. The activities of SOD and catalase were also affected largely by GT consumption in these tissues. The activity of SOD was significantly lower in the intestine and liver but increased in the renal cortex and medulla. In contrast, the activity of catalase profoundly increased in the intestine and liver and to a lesser extent in the renal cortex but decreased in the renal medulla. The present results imply that the biological defense system was perturbed by GT consumption as a result of free radical scavenging properties of its polyphenols and other active constituents. The profound lowering of LPO in the renal cortex and liver in GT compared with control rats suggests that oxidative damage even under normal physiologic conditions was significantly lowered by GT constituents. The reduction in LPO in the liver was associated with a profound increase in catalase activity, whereas in the renal cortex it appeared to be due to increases in catalase and SOD activities. In contrast, an increase in LPO in the intestine was associated with a marked decrease of SOD activity, although there was a significant increase in catalase activity. However, in the renal medulla enhanced LPO can be attributed to decreased catalase activity. The cellular response to oxidative stress in the intestine and renal medulla compared with the liver and renal cortex might have been due to the nature of metabolic activities in these tissues. Although the liver and renal cortex have higher oxidative metabolism, anaerobic metabolism is more prevalent in the intestine and renal medulla due to lower oxygen tension in these tissues. The significant effects of GT consumption on various parameters in different tissues can also be attributed to the presence of active tea components after digestion/absorption/extraction or due to accumulation in various tissues. However, these observations clearly demonstrate that GT consumption activates the antioxidant defense system in all tissues. GT-induced antioxidant defense mechanism in the intestine and liver appeared to be largely catalase mediated, whereas in renal tissues it was predominantly SOD mediated. The activities of certain marker enzymes except LAP in the kidney and ALP in the intestine were also increased significantly in BBMs, which is major target of oxidative damage. It is very likely that the increase in activities of these enzymes was due to protection provided by GT polyphenol-induced lowering of oxidative damage to membranes compared with non-GT-consuming control rats. Conclusions We conclude that the ingestion of GT produced a significant increase in the activities of certain enzymes of glucose degradation and its synthesis in the intestine, liver, and kidney. The effects seem in part to be mediated by GT polyphenols having antioxidant free radical scavenging properties that lower oxidative damage. 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