OBJECTIVE: Obesity has increased at an alarming rate in recent years and is now a worldwide health problem. We investigated the effects of long-term feeding with tea catechins, which are naturally occurring polyphenolic compounds widely consumed in Asian countries, on the development of obesity in C57BL/6J mice.
DESIGN: We measured body weight, adipose tissue mass and liver fat content in mice fed diets containing either low-fat (5% triglyceride (TG)), high-fat (30% TG), or high-fat supplemented with 0.1–0.5% (w/w) tea catechins for 11 months. The β-oxidation activities and related mRNA levels were measured after 1 month of feeding.
RESULTS: Supplementation with tea catechins resulted in a significant reduction of high-fat diet-induced body weight gain, visceral and liver fat accumulation, and the development of hyperinsulinemia and hyperleptinemia. Feeding with tea catechins for 1 month significantly increased acyl-CoA oxidase and medium chain acyl-CoA dehydrogenase mRNA expression as well as β-oxidation activity in the liver.
CONCLUSION: The stimulation of hepatic lipid metabolism might be a factor responsible for the anti-obesity effects of tea catechins. The present results suggest that long-term consumption of tea catechins is beneficial for the suppression of diet-induced obesity, and it may reduce the risk of associated diseases including diabetes and coronary heart disease.
Obesity has increased at an alarming rate in recent years and is now a worldwide health problem. It is widely accepted that obesity results from disequilibrium between energy intake and expenditure, and it is known to be a strong risk factor for type 2 diabetes associated with insulin resistance.1,2
Green tea, one of the most popular beverages consumed in Asian countries, contains a series of polyphenols known as catechins, which consist mainly of epigallocatechin gallate (EGCG), epicatechin gallate and gallocatechin gallate. Tea and tea components have been reported to possess various biological and pharmacological effects, such as antibacterial3 and anticarcinogenic activities,4 and lowering of plasma lipids and glucose levels.5,6 Consequently, interest in the therapeutic applications of tea catechins (or naturally occurring polyphenols) for the treatment and prevention of disease has seen an increase.7
Recent reports by Kao et al showed that intraperitoneal injection of EGCG modulates appetite and reduces food intake through the leptin receptor-independent pathway in rats.8,9 Dulloo et al showed that ingestion of tea catechins stimulated O2 consumption and energy expenditure and decreased the respiratory quotient in humans.10 In addition, green tea extract has been reported to stimulate brown adipose tissue thermogenesis along with fat oxidation, and it was suggested that its thermogenic properties might result primarily from the interaction of its high catechin and caffeine content with sympathetically released noradrenaline.11 Previous reports led us to speculate that dietary tea catechins affect the development of obesity through the modulation of whole-body energy metabolism. However, the effects of long-term intake of tea catechins on diet-induced obesity have not yet been fully determined.
The present study was designed to quantitatively examine the effects of dietary tea catechins on the development of obesity in C57BL/6J mice, which are known as a good model of diet-induced obesity.12 Furthermore, to reveal the underlying mechanisms of action at a molecular level, we examined the effect of tea catechins on the expression of the genes involved in lipid metabolism.
Tea catechins were isolated from green tea (Camellia sinensis) and the composition was determined as described previously.13 Total polyphenol content was 92%, and total catechin content was 73% (w/w). Caffeine content was 0.1%. The composition of isolated tea catechins was epigallocatechin gallate (74%), epicatechin gallate (18%), gallocatechin gallate (6%) and others (2%).
Animals and diets
Male C57BL/6J mice obtained from Japan Clea (Tokyo, Japan) at 7 weeks of age were maintained at 23±1°C under a 12 h light–dark cycle (lights on from 07:00 to 19:00). The mice were fed laboratory chow for 1 week to stabilize their metabolic condition. Mice were divided into five groups (five mice/cage) and were allowed ad libitum access to water and one of the following synthetic diets: a low-fat diet containing 5% (w/w) fat, 20% casein, 66.5% potato starch, 4% cellulose, 3.5% vitamins and 1% minerals; a high-fat diet containing 30% fat, 20% casein, 28.5% starch, 13% sucrose, 4% cellulose, 3.5% vitamins and 1% minerals; a tea catechins diet, which consisted of the high-fat diet supplemented with the indicated amount of tea catechins. Animals were maintained on these diets for 11 months. The fatty acid composition of fat was 46.7% linoleic acid, 36.0% oleic acid, 7.5% α-linolenic acid and 5.7% palmitic acid.
Mice were divided into three groups (five mice/cage) and were allowed ad libitum access to their respective synthetic diets for 1 month. On the final day of the experiment, mice in all groups were killed, and each tissue sample was rapidly dissected for β-oxidation assay and Northern blot analysis.
Body weight and food intake
Body weight was measured weekly throughout the study. Food intake was measured on a per-cage basis over the course of 24 h one day per week.
Measurement of fecal lipids
Feces were collected four times on a per-cage basis for a 24 h period after 3–4 weeks of feeding. After drying, total lipids of feces were extracted by the procedure of Folch et al,14 and the amount of extracted lipid was determined gravimetrically.
On the final day of the experiments, blood was collected via the post-caval vein from anesthetized animals (non-fasting condition). Blood was collected under the fasting condition by cutting the tail at 10 days prior to the end of the experiment. Plasma analyses were conducted as described previously.15
Measurement of liver triglyceride and cholesterol
β-Oxidation activity was measured as previously reported,16 with minor modification. Frozen mouse liver, skeletal muscle (gastrocnemius and soleus), interscapular brown adipose tissue (BAT), and small intestinal mucosa were thawed and homogenized on ice with 5 vols of 250 mM sucrose containing 1 mM EDTA and 10 mM HEPES (pH 7.2), and centrifuged at 600 g for 5 min. The resultant supernatant was used for the assay. The reaction mixture contained Dulbecco's phosphate-buffered saline (pH 7.2), 1 mM ADP, 1 mM MgCl2, 0.25 mM CoA, 1 mM L-carnitine, 0.5 mM L-malic acid, 1 mM DTT, 0.2 mg/ml BSA, 0.1 µCi [14C]-palmitic acid, and the extract containing 100 µg protein, in a final volume of 200 µl. The reaction was started by adding the substrate and incubating the preparation at 37°C for 25 min. The reaction was terminated by adding 200 µl of 0.6 N perchloric acid, followed by centrifugation. The supernatant was extracted three times with 800 µl of n-hexane to remove residual radiolabeled palmitate. Radioactivity of the water phase was measured. Protein concentrations were determined using a DC protein assay kit (Bio Rad, Hercules, CA, USA).
RNA extraction and Northern blot analysis
On the final day of Experiment 2, mice were killed between 9:00 and 11:00, and the liver was rapidly dissected and subjected to RNA extraction. Total RNA was isolated using Isogen (Wako) according to the manufacturer's instructions. Purified RNA (20 µg) was electrophoresed on 1% agarose/ formamide gels and blotted onto Hybond-N+ membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blotted membranes were hybridized with a 32P-labeled cDNA probe at 42°C overnight. After washing, membranes were autoradiographed and analyzed with a BAS2000 bioimage analyzer (Fuji Photo Film, Tokyo, Japan). The membranes were also hybridized with a 32 P-labeled 36B4 probe, and the mRNA levels were calculated relative to the 36B4 mRNA levels. Normalized values were expressed as percentages, using the value of mice fed a low-fat diet as 100%. The PCR-generated cDNA probes were as follows: ACO (Genbank AF006688, nt 218-880), MCAD (J02791, nt 671-1199), FAS (X13135, nt 336-1483) and 36B4 (X15267, nt 97-860).
All values are presented as mean±s.d. Statistical comparisons of the groups were made by ANOVA, and each group was compared with the others by Fisher's PLSD test (StatView, SAS Institute Inc., NC, USA). Statistical significance was defined as P<0.05.
Body weight, food intake and visceral fat accumulation
As shown in Figure 1A, high-fat diet-induced body weight gain was markedly reduced by feeding with diets containing 0.2 and 0.5% (w/w) tea catechins. Although no significant differences were observed in average energy intake between the high-fat-fed mice and the 0.1 or 0.2% tea catechins-fed mice, energy intake in the 0.5% tea catechins-fed mice was lowered by 5.6% (Table 1). Compared with that of mice fed the high-fat diet, the body weight in mice fed 0.5% tea catechins was significantly lower after 12 weeks of feeding (P<0.05), and during that period the cumulative energy intake values between the high-fat and the 0.5% tea catechins group were nearly identical (Figure 1B). The body weight in mice fed 0.2% tea catechins was significantly lowered after 27 weeks of feeding (P<0.05).
To examine the effects of tea catechins on visceral fat accumulation, we analyzed the distribution of fat in three individual fat pads. Supplementation with tea catechins significantly suppressed epididymal, retroperitoneal and perirenal fat accumulation, in comparison with that in the high-fat diet group, in a dose-dependent manner (Table 1). These results suggested that feeding of tea catechins is useful for the suppression of body fat accumulation.
Liver lipid accumulation
The liver weight and lipid contents are shown in Table 1. Compared with the low-fat diet, feeding with the high-fat diet resulted in a significant increase in liver TG content. However, the TG contents in 0.2 and 0.5% tea catechins-fed mice were significantly lower than that in the high-fat-fed mice. Note that we found no significant difference in liver cholesterol content among these experimental groups.
In accordance with previous studies,12,15 in mice fed the high-fat diet, plasma insulin and leptin levels under fasting and non-fasting conditions were significantly higher than those in low-fat-fed mice (Table 2), suggesting the development of hyperinsulinemia and hyperleptinemia during the experimental period. However, in contrast to high-fat-fed mice, those receiving supplementation with 0.2 and 0.5% tea catechins showed significantly suppressed elevation of plasma insulin and leptin concentrations.
β-Oxidation activities in various tissues
To elucidate the mechanisms underlying the beneficial effects of tea catechins, we examined fatty acid β-oxidation activity in various tissues; however, it was difficult to determine whether the changes in lipid metabolism observed in this study were a primary cause or a secondary consequence of the reduced body fat accumulation. Therefore, we investigated β-oxidation activity after 1 month of feeding (Experiment 2), at which time no significant differences were observed in food intake or body weight among the high-fat groups (data not shown). Among the organs examined, the most marked changes were observed in the liver. As shown in Figure 2, tea catechins significantly increased β-oxidation activity by 285 and 180% over those of the low-fat and high-fat diet group, respectively, indicating that consumption of tea catechins stimulated lipid catabolism in the liver. In contrast, no significant difference was observed in β-oxidation activity in the small intestine, BAT and the skeletal muscle between mice fed high-fat and tea catechins diets.
mRNA expression in the liver
We analyzed the mRNA levels of genes involved in lipid metabolism in the liver by Northern blotting (Experiment 2). Supplementation with tea catechins significantly up-regulated mRNA expression of acyl-CoA oxidase (ACO), which is a peroxisomal β-oxidation enzyme, and medium-chain acyl-CoA dehydrogenase (MCAD), which is a mitochondrial β-oxidation enzyme in the liver, in comparison with levels in the high-fat diet group (Figure 3). On the other hand, the levels of fatty acid synthase (FAS) mRNA were lowered by high-fat diets, and no significant change was observed following supplementation with tea catechins.
In this study, we examined the effects of tea catechins on the development of obesity in C57BL/6J mice. The present results clearly showed that the feeding of tea catechins is beneficial for the suppression of diet-induced obesity. We confirmed that the doses of tea catechins (0.1–0.5%) used in this study did not increase the fecal excretion of lipids (Table 1). In addition, no significant differences were observed in average energy intake between the high-fat-fed mice and the 0.1–0.2% tea catechins-fed mice. Energy intake in 0.5% tea catechins-fed mice was slightly lowered; however, it was difficult to determine whether the decrease in energy intake was a cause of the reduced body fat accumulation. Body weight in 0.5% tea catechins-fed mice was significantly lowered as early as 12 weeks after feeding the diet supplemented with tea catechins, with no significant decrease in energy intake (Figure 1), indicating that the anti-obesity effect of tea catechins could not be attributed solely to the decrease in energy intake. Rather, some additional mechanisms leading to energy expenditure might be involved in the anti-obesity effect of tea catechins. These findings suggesting that tea catechins potently activate β-oxidation of fatty acid in the liver may indicate an underlying mechanism of the beneficial effects of tea catechins. Considering the absorption and behavior of catechins, it appears reasonable that they would influence the liver. Previous studies on the metabolism of catechins have demonstrated that ingested catechins are absorbed through the intestinal tract, and high levels of catechins were detected in the liver,17,18 suggesting that this organ is susceptible to dietary tea catechins. Since the liver is known as an organ active in β-oxidation,19,20 up-regulation of hepatic lipid metabolism may contribute to the suppression of liver fat and visceral fat accumulation. On the other hand, the mRNA level of FAS was not altered by tea catechins, which may mean that the stimulation of fatty acid oxidation, rather than the suppression of lipogenesis, is the predominant contribution among the effects of tea catechins.
We recently demonstrated that long-term intake of tea catechins at 600 mg/day decreased visceral fat mass, as determined by computed tomography in humans.21 Assuming that an infusion of green tea contains 1 g/l catechins,22 daily intake of 600 ml of green tea is expected to reduce body weight similarly to visceral fat.
The administration of green tea extract is reported to increase fat oxidation and energy expenditure in humans10 and in rat brown adipose tissue,11 and it has been assumed to be attributable to an interaction between green tea extract's high catechins and caffeine content that influences the level of sympathetic activity. Since catechins are known to inhibit catechol-O-methyltransferase (the enzyme that degrades noradrenaline), and caffeine is known to inhibit the phosphodiesterase-induced degradation of cAMP, it has been proposed that these compounds synergistically prolong and augment the sympathetic stimulation of fat oxidation.11 However, the tea catechins used in the present study contain much less caffeine than did the tea extracts used in previous studies; therefore, the stimulatory effect of tea catechins on lipid metabolism is probably attributable to the effect of catechins, and not to that of caffeine per se. In contrast to the present findings that tea catechins enhanced the hepatic β-oxidation accompanying related gene expression, the stimulation of energy expenditure and fat oxidation by the combination of catechins and caffeine found in previous studies was a relatively acute response. We have confirmed that up-regulation of hepatic β-oxidation enzymes did not occur within 24 h after feeding with tea catechins (data not shown). Therefore, the mechanism of tea catechins observed in the present study may differ from those of catechins in the presence of caffeine. These findings, taken together, suggest that a single administration of green tea rich in catechins and caffeine may stimulate fat oxidation via the sympathetic system, and long-term administration (eg via diet) may further stimulate fat oxidation trough up-regulation of the β-oxidation pathway. Thus, tea catechins have the potential to influence body fat accumulation.
The precise molecular mechanism by which tea catechins stimulate lipid metabolism is unclear at present. In the last decade, it has become apparent that the expression of many lipid-metabolizing enzymes, including ACO and MCAD, is transcriptionally regulated by peroxisome proliferator-activated receptors (PPARs).23 We confirmed that a series of catechins (EGCG, ECG, GCG, etc) are not ligands for PPARα by using a transient transfection assay that combines a chimeric GAL4 DNA binding domain (DBD)-PPARα ligand binding domain (LBD) receptor and a reporter gene containing GAL4 binding sites (data not shown). On the other hand, nuclear factor-κB (NF-κB) was reported to inhibit PPARα-mediated activation of a PPAR response element-driven promoter through physical interaction of PPARα with NF-κB p65.24 Because catechin gallates inhibit the activation of NF-κB,25,26 feeding with tea catechins regulates the transcription of PPAR-related genes by reducing the NF-κB activation, which may lead to up-regulation of the lipid-metabolizing enzymes. Further studies are necessary to elucidate the transcriptional regulatory mechanisms involved in tea catechins-induced gene expression.
In summary, we demonstrated that long-term feeding of tea catechins is beneficial for the suppression of high-fat diet-induced obesity, and that their effects may be attributed, at least in part, to the activation of lipid metabolism in the liver. These results suggest that a sufficient supply of tea catechins may prevent or improve obesity by modulating lipid metabolism and possibly reduce the risk of associated diseases, including diabetes and coronary heart disease.
Larsson B, Bjorntorp P, Tibblin G . The health consequences of moderate obesity Int J Obes 1981 5: 97–116.
Hartz AJ, Rupley DC Jr, Kalkhoff RD, Rimm AA . Relationship of obesity to diabetes: influence of obesity level and body fat distribution Prev Med 1983 12: 351–357.
Shiota S, Shimizu M, Mizushima T, Ito H, Hatano T, Yoshida T, Tsuchiya T . Marked reduction in the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus produced by epicatechin gallate, an ingredient of green tea (Camellia sinensis) Biol Pharm Bull 1999 22: 1388–1390.
Yang CS, Wang ZY . Tea and cancer J Natl Cancer Inst 1993 85: 1038–1049.
Muramatsu K, Fukuyo M, Hara Y . Effect of green tea catechins on plasma cholesterol level in cholesterol-fed rats J Nutr Sci Vitaminol 1986 32: 613–622.
Matsumoto N, Ishigaki F, Ishigaki A, Iwashima H, Hara Y . Reduction of blood glucose levels by tea catechin Biosci Biotech Biochem 1993 57: 525–527.
Mukhtar H, Ahmad N . Tea polyphenols: prevention of cancer and optimizing health Am J Clin Nutr 2000 71 (6 Suppl): 1698S–1702S.
Kao YH, Hiipakka RA, Liao S . Modulation of endocrine systems and food intake by green. tea epigallocatechin gallate Endocrinology 2000 141: 980–987.
Kao YH, Hiipakka RA, Liao S . Modulation of obesity by a green tea catechin Am J Clin Nutr 2000 72: 1232–1234.
Dulloo AG, Duret C, Rohrer D, Girardier L, Mensi N, Fathi M, Chantre P, Vandermander J . Efficacy of a green tea extract rich in catechin polyphenols and caffeine in increasing 24-h energy expenditure and fat oxidation in humans Am J Clin Nutr 1999 70: 1040–1045.
Dulloo AG, Seydoux J, Girardier L, Chantre P, Vandermander J . Green tea and thermogenesis: interactions between catechin-polyphenols, caffeine and sympathetic activity Int J Obes Relat Metab Disord 2000 24: 252–258.
Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN . Diet-induced type II diabetes in C57BL/6J mice Diabetes 1988 37: 1163–1167.
Goto T, Yoshida Y, Kiso M, Nagashima H . Simultaneous analysis of individual catechins and caffeine in green tea J Chromatogr 1996 749: 295–299.
Folch J, Leens M, Sloane-Stanley GH . A simple method for the isolation and purification of total lipids from animal tissue J Biol Chem 1957 226: 497–509.
Murase T, Mizuno T, Omachi T, Onizawa K, Komine Y, Kondo H, Hase T, Tokimitsu I . Dietary diacylglycerol suppresses high-fat and high sucrose diet-induced body fat accumulation in C57BL/6J mice J Lipid Res 2001 42: 372–378.
Singh H, Beckman K, Poulos A . Peroxisomal β-oxidation of branched chain fatty acids in rat liver. Evidence that carnitine palmitoyltransferase I prevents transport of branched chain fatty acids into mitochondria J Biol Chem 1994 269: 9514–9520.
Nakagawa K, Miyazawa T . Absorption and distribution of tea catechin, (−)-epigallocatechin-3-gallate, in the rat J Nutr Sci Vitaminol 1997 43: 679–684.
Suganuma M, Okabe S, Oniyama M, Tada Y, Ito H, Fujiki H . Wide distribution of [3H](−)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue Carcinogenesis 1998 19: 1771–1776.
Nemali MR, Usuda N, Reddy MK, Oyasu K, Hashimoto T, Osumi T, Rao MS, Reddy JK . Comparison of constitutive and inducible levels of expression of peroxisomal beta-oxidation and catalase genes in liver and extrahepatic tissues of rat Cancer Res 1988 48: 5316–5324.
Kelly DP, Gordon JI, Alpers R, Strauss AW . The tissue-specific expression and developmental regulation of two nuclear genes encoding rat mitochondrial proteins J Biol Chem 1989 264: 18921–18925.
Nagao T, Meguro S, Soga S, Otsuka A, Tomonobu K, Fumoto S, Chikama A, Mori K, Yuzawa M, Watanabe H, Hase T, Tanaka Y, Tokimitsu I, Shimasaki H, Itakura H . Tea catechins suppress accumulation of body fat in humans J Oleo Sci 2001 50: 717–728.
Scalbert A, Williamson G . Dietary intake and bioavailability of polyphenols J Nutr 2000 130: 2073S–2085S.
Schoonjans K, Staels B, Auwerx J . Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression J Lipid Res 1996 37: 907–925.
Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G, Staels B . Peroxisome proliferator-activated receptor α negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-κB and AP-1 J Biol Chem 1999 274: 32048–32054.
Lin YL, Lin JK . (−)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-κB Mol Pharmac 1997 52: 465–472.
Murase T, Kume N, Hase T, Shibuya Y, Nishizawa Y, Tokimitsu I, Kita T . Gallates inhibit cytokine-induced nuclear translocation of NE-κB and expression of leukocyte adhesion molecules in vascular endothelial cells Arterioscler Thromb Vasc Biol 1999 19: 1412–1420.
About this article
The Combination of ‘Benifuuki’ with Quercetin Suppresses Hepatic Fat Accumulation in High-Fat High-Cholesterol Diet-Fed Rats
Journal of Nutritional Science and Vitaminology (2019)
Dietary supplement of Smilax china L. ethanol extract alleviates the lipid accumulation by activating AMPK pathways in high-fat diet fed mice
Nutrition & Metabolism (2019)
Prosopis alba seed flour improves vascular function in a rabbit model of high fat diet-induced metabolic syndrome