(−)-Epigallocatechin-3-gallate inhibits pancreatic lipase and reduces body weight gain in High Fat-Fed Obese Mice

Abstract

Tea (Camellia sinensis,Theaceae) has been shown to have obesity preventive effects in laboratory studies. We hypothesized that dietary epigallocatechin-3-gallate (EGCG) could reverse metabolic syndrome in high fat-fed obese C57BL/6J mice, and that these effects were related to inhibition of pancreatic lipase (PL). Following treatment with 0.32% EGCG for 6 weeks, a 44% decrease in body weight gain in high fat-fed, obese mice (p < 0.01) was observed compared to controls. EGCG treatment increased fecal lipid content by 29.4% (p < 0.05) compared to high fat fed control, whereas in vitro, EGCG dose-dependently inhibited PL (IC₅₀ = 7.5 μM) in a non-competitive manner with respect to substrate concentration. (−)-epicatechin-3-gallate exhibited similar inhibitory activity, whereas the non-ester-containing (−)-epigallocatechin did not. In conclusion, EGCG supplementation reduced final body weight and body weight gain in obese mice, and some of these effects may be due to inhibition of PL by EGCG.

INTRODUCTION

Tea (Camellia sinensis) is the second most popular beverage in the world behind water. Several studies have shown that green tea, and its major polyphenolic component, (−)-epigallocatechin-3-gallate (EGCG), may modulate body weight and fat (reviewed in (1)). A limited number of human intervention studies have examined the effect of green tea supplementation on obese subjects (2, 3). For example, treatment of obese Japanese subjects with green tea per day for 12 weeks significantly decreased body weight and body mass index compared to placebo (4). A larger number of studies in animal models have demonstrated the efficacy of tea polyphenols for the prevention of obesity and obesity-related pathologies (1). For example, Wolfram et. al., found that supplementation of high fat fed mice with 1% (w/w) dietary TEAVIGO (94% EGCG) for 5 months reduced increases in body weight, fed state glucose levels, triglycerides and leptin (5). Treatment with green tea extract reduced body weight in leptin-deficient mice (ob/ob) (6). Few studies have shown that green tea can reverse symptoms related to obesity. Lee et. al., found that high fat-fed obese mice treated with 0.5% EGCG for 8 weeks had decreased body weight compared to the high fat fed controls (7). Some studies have observed that EGCG and green tea can modulate the expression of genes related to lipid catabolism and biogenesis, although, the efficacy and underlying mechanisms of action of EGCG to reverse obesity needs to be further investigated (reviewed in (1)). Pancreatic lipase (PL) is an enzyme secreted into the duodenum that plays a key role in digestion and absorption of fats and is a target for intervention (8,9). The enzyme first rapidly hydrolyzes triglycerides to diglycerides, and then slowly to a monoglyceride (10). Ikeda et al., studied the effect of green tea catechins against PL but found relatively weak inhibition (effective concentrations greater than 1 mM) (11). Although interesting, the study was limited and did not examine the mechanism of enzyme inhibition. Here, we examined the ability of EGCG to modulate fat absorption and body weight gain in high fat-fed obese mice and sought to gain insight into the mechanisms of action.

METHODS AND PROCEDURES

Chemicals and diet

EGCG (93% pure) was purchased from Taiyo Green Power Company (Jiangsu, China). Lipase (type II, from porcine pancreas), 4-nitrophenyl butyrate (4-NPB), orlistat, (−)-epicatechin-3-gallate (ECG, 98% pure) and (−)-epigallocatechin (EGC, >95% pure) were purchased from Sigma Chemical Company (St. Louis, MO). Diets were prepared by Research Diets, Inc. (New Brunswick, NJ) and have been previously described (12).

Treatment and analysis

Male C57bl/6J mice, 5 weeks old from Jackson Laboratories (Bar Harbor, ME) were maintained on 12 h light/dark and had access to food and water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University (IACUC #28962). Mice were maintained on high fat diet (60% kcal fat) for 9 weeks and then divided into 2 treatment groups. The first was continued on high fat diet (HF) and the second was switched to high fat diet supplemented with 0.32% EGCG (HF-HFE). The experiment was continued for an additional 6 weeks. Feces (24 h total cage sample) were collected at week 10, 12 and 14 of the study.

Fecal samples were solubilized in deionized water (1:2, w:v) overnight at 4°C. The samples were vortexed and extracted with methanol:chloroform (2:1, v:v). The organic phase was filtered, dried under vacuum, weighed and normalized to fecal weight.

PL was suspended in water (10 mg/mL). The solution was then centrifuged for 10 minutes at 1050 g and the supernatant was used as the enzyme source for the experiment. For each experiment, 0.1 mg PL and the test inhibitor were combined in 10 mM Tris-HCl (pH=8.0). The reaction was started by addition of 4-NPB (final concentration of 190 μM). The tube was vortexed for 10 s, incubated at room temperature for 10 min, and the conversion of 4-NPB to 4-nitrophenol (4-NP) was monitored at 400 nm using a spectrophotometer.

Enzyme inhibition kinetic analysis was performed by holding inhibitor concentration constant and using 6 different concentrations of 4-NPB (0 – 0.50 mM) in a manner analogous to above. The maximum velocity (V_max) and Michaelis-Menten constant (K_m) were determined by fitting the initial velocity data at each concentration of 4-NPB.

Statistics

All plots show the mean ± standard error of the mean (SEM). Student’s t test was used to compare body weight (BW) gain. Differences in K_m and V_max were tested by One-way ANOVA with Tukey’s post-test. Two-way ANOVA with Bonferroni’s post-test was used for BW and food consumption. Statistical significance was achieved at p < 0.05.

RESULTS

Throughout the study, the food intake did not differ between groups (data not shown). At week 9, both HF and HF-HFE mice had a mean body weight of 40.3 g. After 6 weeks of EGCG treatment, HF-HFE mice weighed 5.4% less than the HF control (Fig. 1A, p < 0.05). EGCG treatment reduced body weight gain from 2.2 ± 0.1 g/wk (HF) to 1.3 ± 0.1 g/wk (HF-HFE) (p < 0.05).

Figure 1.

EGCG modulates body weight and fat digestion. The effect of EGCG on (A) body weight gain and (B) fecal lipid content was determined in high fat-fed obese mice. High fat-fed C57bl/6J mice were treated with EGCG (HF-HFE) for 7 weeks and body weight was determined weekly. Values in the graph represent the mean. Error bars were omitted for clarity. * indicates a statistically significantly difference (p < 0.05) from HF mice by two-way ANOVA with Bonferroni’s post-test (for body weight) or Student’s t test (for body weight gain). (C) The inhibitory effect of the tea catechins against PL in vitro was determined. (D) Kinetic analysis was performed to understand the mechanism by which EGCG inhibits PL in vitro. The symbols represent the mean of four independent experiments done in duplicate. Error bars represent the SEM.

EGCG treatment increased mean fecal lipid content by 29.4% compared to high fat-fed obese controls (Fig. 1B, p < 0.05). PL represents a potential mechanistic target to explain increased fecal lipid content. EGCG dose-dependently inhibited PL with 50% inhibition at 7.5 μM (Fig. 1C). Above 7.5 μM, no further increase in inhibition was observed. ECG, which retains a galloyl ester, had a maximum inhibition of 40% at 5 μM. By contrast, EGC, which lacks a galloyl ester, had no significant inhibitory effect at concentrations up to 100 μM. The inhibitory kinetics of EGCG were determined with respect to 4-NPB concentration (Fig. 1D). Analysis of the Michaelis-Menten plot showed that V_max decreased from 14.6 ± 0.7 pmol/min/mg to 7.8 ± 0.6 pmol/min/mg as the concentration of EGCG increased from 0 μM to 5 μM (p < 0.05). A trend for increasing K_m was observed as a function of EGCG concentration (128.1 ± 26.1 μM at 5 μM EGCG vs. 116.7 ± 16.2 μM at 0 μM), but was not significant. These results suggest that EGCG noncompetitively inhibits PL with respect to substrate concentration. Orlistat inhibited PL with an IC₅₀ of 0.5 μM (Fig. 1D).

DISCUSSION

In the present study, ECGC treatment of obese mice for 6 weeks reduced body weight gain by 44% compared to HF treated mice. These results are similar to those of Lee et al., who reported a decrease in body weight of obese mice after 8 weeks of treatment with EGCG (7). Bose et al., did not find a significant difference in body weight with EGCG supplementation compared to high fat control (12). These results suggest that continued treatment of obese mice with EGCG may enhance changes in body weight gain. EGCG treatment caused a 29.4% increase in mean fecal lipid levels compared to HF mice. This increase suggests that EGCG decreases lipid absorption. Previous studies have examined the effect of EGCG on fecal lipid content following long-term treatment of lean mice, and correlated those effects to prevention of obesity (12). To our knowledge, this is the first demonstration of EGCG-induced changes in fecal lipid output by obese mice.

Pancreatic lipase is a key enzyme in fat digestion, and PL inhibition is associated with decreased fat absorption. We found that EGCG potently inhibited PL activity in vitro (IC₅₀ = 7.5 μM). Nakai et al., found similar inhibition, but the IC₅₀ values are significantly different. This may be due to use of a different enzyme sources or different substrates (13). We report for the first time that EGCG inhibited PL in a non-competitive manner with regard to substrate concentration. This is an interesting observation given the apparent requirement of the galloyl ester for activity. These results suggest that EGCG may modify the active site of PL irreversibly, perhaps through some oxidative interactions with cysteine residues. Such mechanism is speculative and will require additional studies.

The effective concentrations of EGCG against PL are comparable to those achievable in vivo. After oral ingestion of 75 mg/kg EGCG, the peak concentration of EGCG in mouse small intestine was reported to be 20.9 μg/g (14). The concentration found in the intestinal contents is likely much higher.

Although the present effects on lipid absorption and PL are interesting, the increase in fecal lipid content is relatively small and likely does not account for the total change in body weight gain observed. Other mechanisms related to fatty acid oxidation and de novo lipogenesis may also be involved. Indeed, previous studies have shown that EGCG and green tea can modulate the processes in liver, adipose tissue, and skeletal muscle (reviewed in (1)). Further studies are needed to determine the relative importance of these various mechanisms in human subjects.

In conclusion, treatment of high fat-fed obese mice with 0.32% EGCG can modulate body weight gain, and these effects appear to be due in part to EGCG-mediated modulation of lipid absorption possibly via PL. These results should be confirmed by additional animal studies and short-term intervention studies in obese human subjects.

Abbreviations:

4-NP

4-nitrophenol

4-NPB

4-nitrophenyl butyrate

B.W.

body weight

ECG

(−)-epicatechin-3-gallate

EGC

(−)-epigallocatechin

EGCG

(−)-epigallocatechin-3-gallate

GTE

green tea extract

HF

high fat-diet fed mice

HF-HFE

high fat diet fed mice treated with EGCG

IC₅₀

median inhibitory concentration

K_m

Michaelis-Menten constant

ORFLD

obesity-related fatty liver disease

PL

pancreatic lipase

V_max

maximum velocity