Oral Green Tea Catechins Transiently Lower Plasma Glucose Concentrations in Female db/db Mice

Abstract

Polyphenols, including green tea catechins, are secondary plant compounds often discussed in the context of health-promoting potential. Evidence for such effects is mainly derived from epidemiological and cell culture studies. The aim of the present study was to investigate antidiabetic, antiadipogenic, and anti-inflammatory effects at nonpharmacological doses in an obese diabetic mouse model that exerts early relevant clinical signs of non–insulin-dependent diabetes mellitus. Female db/db mice received a flavonoid-poor diet either without additive, with rosiglitazone (RSG, 0.02 g/kg diet), or with green tea extract (low-dose green tea extract [LGTE] and high-dose green tea extract [HGTE], 0.1 and 1 g/kg diet). Food and water were freely available. The body weight was monitored weekly. Blood was sampled (12-h fasted) from the tail vein on day 28 and analyzed for glucose, cholesterol, triacylglycerol, nonesterified fatty acids, insulin, adiponectin, and soluble intercellular adhesion molecule-1 (sICAM-1). Blood glucose was also analyzed on day 14. Furthermore, sICAM-1 release was investigated in tumor necrosis factor alpha-stimulated EAhy926 cells. After 14 days, fasting glycemia was improved by RSG or HGTE supplementation compared to controls. However, at the end of the study (day 28), only RSG exhibited glucose-lowering effects and induced plasma adiponectin concentrations, paralleled by higher body weight gain and reduced periuterine fat pads compared to controls. However, only GTE treatment reduced sICAM-1 release in vitro and in vivo. Nonpharmacological HGTE supplementation in db/db mice caused (1) no adiponectin-inducing or antiadipogenic effects, (2) reduced sICAM-1 release, thereby potentially exerting anti-inflammatory effects in the progressive diabetic state, and (3) a transient improvement in glycemia.

Introduction

N on–insulin-dependent diabetes mellitus (NIDDM) is a chronic disease that comprises 90% of people with diabetes worldwide and is largely the result of excess body weight.¹ The incidence of obesity paralleled by NIDDM has increased dramatically in recent decades, and a worldwide increase of up to 370 million diabetic individuals is estimated for the year 2030.² Thiazolidinediones improve hypoadiponectinemia and insulin sensitivity in peripheral tissues, reduce hyperglycemia, hyperinsulinemia, and the production of inflammatory cytokines, and are used as potent oral antidiabetics in NIDDM.³ These effects are mainly attributable to their peroxisome proliferator–activated receptor-gamma (PPAR-γ) agonist activity. The PPAR-γ receptor, however, not only is a major target for insulin-sensitizing agents, but also plays an important role in adipocyte differentiation and proliferation.⁴ However, drugs used for the treatment of diabetes are often associated with serious side effects,⁵ and the use of complementary and alternative medicine has tremendously increased in recent years.⁶ Plant-derived flavonoids have long been recognized to possess health-promoting effects.^7,8 Green tea is the major source of catechins (Fig. 1), also called flavan-3-ols or flavans, which are a subgroup of flavonoids.⁹ Regular green tea consumption has been associated with a health-supporting lifestyle.¹⁰ Effects of green tea catechins are mainly related to its epigallocatechin-3-gallate (EGCG) content¹¹ and include antiobesity,¹⁰ antidiabetic,¹² anti-inflammatory,¹³ and antioxidative¹⁴ properties. Biological effects of EGCG have been attributed to the following mechanisms: inhibition of adipocyte differentiation/proliferation,¹⁵ inhibition of tumor necrosis factor alpha (TNF-α)–induced signaling,¹⁶ elevation of circulating adiponectin concentrations,¹⁷ and enhanced tissue glucose uptake.¹⁸ Although epidemiological and cell culture-derived results strongly indicate health-promoting effects of green tea extract or EGCG, evidence from experimental animal and human studies remains inconclusive. Thus, the aim of this study was to investigate possible antiobesity, antidiabetic, and anti-inflammatory effects of a nonpharmacological green tea extract supplementation in an obese, insulin-resistant mouse model. The db/db mouse becomes markedly obese by 6 weeks of age accompanied by fasting hyperglycemia, hyperinsulinemia, and hypoadiponectinemia, indicating that db/db mice are not insulin deficient, but instead display distinct signs of insulin resistance.¹⁹

FIG. 1.

Plasma glucose concentrations of db/db mice in the 12-h fasted state after 14 and 28 days of feeding either without additive (control [C]) or with the addition of rosiglitazone (RSG, 0.02 g/kg diet) or green tea extract (0.1 or 1 g/kg diet, low-dose green tea extract [LGTE] and high-dose green tea extract [HGTE]). Data are means+standard error of the mean (SEM; n=8), two-way analysis of variance (ANOVA), *P<.05 compared to controls.

Materials And Methods

Animals and diets

Female db/db mice (BKS.Cg-m+/+Lepr<db>/J; n=32), initial body weight 30.1±0.5 g (Charles River Laboratories, Brussels, Belgium), were housed in cages with sawdust-covered solid flooring in a controlled environment (22°C±2°C, humidity 65%) with a 12-h light/dark cycle. Animals had free access to food and tap water. During the first week, all animals were fed a flavonoid-poor diet (C1000 control diet; Altromin GmbH, Lage, Germany; Table 1) without additives; thereafter, animals were randomly assigned to one of four groups and received the same flavonoid-poor diet either without additive (C), with the addition of rosiglitazone (RSG, 0.02 g/kg diet; Avandia^®, GlaxoSmithKline, Munich, Germany) or green tea extract (0.1 or 1 g/kg diet, low-dose green tea extract [LGTE] and high-dose green tea extract [HGTE]; Polyphenon 60, Sigma-Aldrich Chemie GmbH, Steinheim, Germany, Table 2) for 28 days. Diets were prepared daily, and supplement-added, just before, feed was offered (daily at the beginning of the dark phase after removal of feed residues). The body weight was monitored weekly. Blood was collected from the tail vein for fasting (12 h) glucose measurements (HemoCue^® System; HemoCue GmbH, Grossostheim, Germany) on days 14 and 28. After the 28-day feeding period, at the end of the dark phase and the 12-h fasting phase, blood from the tail vein was also collected in EDTA tubes (2.6 mL; Sarstedt AG & Co., Nürnbrecht, Germany) for plasma preparations. Thereafter, animals were sacrificed under anesthesia (CO₂) by decapitation, and all tissues and organs were immediately dissected and snap-frozen in liquid nitrogen for storage at −86°C. Fresh weight of the liver and periuterine adipose tissue was determined before freezing. Animal experiments were approved by the Ministry of Agriculture, the Environment, and Rural Areas of Land Schleswig-Holstein, Germany (No. V 312-72241.121-25).

Table 1.

Crude Nutrient Composition of the Flavonoid-Poor Diet

Content ^a	Amount
Crude protein, %	17.6
Crude fat, %	5.1
Crude fiber, %	4.1
Crude ash, %	5.5
Disaccharide, %	11.1
Polysaccharide, %	47.2
Dry matter, %	91.8
Metabolizable energy, MJ/kg	14.6

Information as provided by the manufacturer (Altromin GmbH).

Table 2.

Polyphenol Content of the Green Tea Extract, Polyphenon 60

Substance	Content ^a (% of dry matter)
Epigallocatechin	16.7
Epigallocatechin gallate	34.0
Epicatechin gallate	8.7
Gallocatechin gallate	2.8
Epicatechin	7.3
Catechin gallate	0.5
Total catechins	70.0

Information as provided by the manufacturer (Sigma-Aldrich Chemie GmbH).

Determination of plasma metabolites and hormones

Plasma triacylglycerols (TG), nonesterified fatty acids (NEFA), and total cholesterol were quantified spectrophotometrically (Konelab 20i; Konelab Corporation, Espoo, Finland) using commercially available kits (TG and cholesterol: Thermo Fisher Scientific GmbH, Dreieich, Germany; NEFA: Wako Chemicals, Neuss, Germany). Plasma concentrations of insulin, adiponectin, and soluble intercellular adhesion molecule-1 (sICAM-1) were measured by enzyme-linked immunosorbent assays (ELISA) using commercial kits (insulin: ELISA Kit; DRG Diagnostics, Marburg, Germany; adiponectin: ELISA Quantikine^® mouse Adiponectin/Acrp30; R&D Systems GmbH, Wiesbaden, Germany; sICAM-1: Quantikine^® mouse ELISA sICAM-1; R&D Systems GmbH).

Cell culture

EAhy926 endothelial-like cells were kindly provided by Dr. Edgell (University of North Carolina, Chapel Hill, NC, USA) and cultured in the Dulbecco's modified Eagle's medium containing glucose (4 g/L), supplemented with l-glutamine (2 mmol/L), fetal bovine serum (10%), penicillin (100 mU/mL), and streptomycin (100 μg/mL). This cell line was obtained by fusion of human umbilical vein endothelial cells with the permanent human lung carcinoma cells A549 and possesses characteristics of endothelial cells. Cells were grown in a humidified incubator at 37°C with 5% CO₂. Confluent cells were split using a trypsin/EDTA mix.

sICAM-1 release in EAhy926 cells

For determination of sICAM-1 in TNF-α-stimulated endothelial cells, 200,000 EAhy926 cells per well were seeded in a 12-well plate. After 24-h cultivation, the culture medium was replaced with a medium containing GTE (10 μg/mL), EGCG (25 μmol/L), or RSG (1 μmol/L; Biozol Diagnostics, Eching, Germany). This RSG concentration was proven to activate PPAR-γ in a reporter gene assay using dimethyl sulfoxide (0.1%) as a negative control. We confirmed that these GTE, EGCG, and RSG concentrations had no adverse effects on cell viability (determined by neutral-red assay). After 24 h of incubation, the medium was aspirated, and the cells were washed once with prewarmed PBS to completely remove test substances. Subsequently, PBS was replaced, and cells were stimulated for 24 h with 1 ng/mL human TNF-α (Invitrogen GmbH, Karlsruhe, Germany). Thereafter, supernatants were collected and aliquots stored at −80°C for sICAM-1 analysis. Supernatant sICAM-1 concentrations were assessed by commercial sandwich ELISA (Quantikine Human ELISA sICAM-1/CD54; R&D Systems GmbH). The undiluted samples were applied to a 96-well plate with immobilized monoclonal antibodies against human sICAM-1. ELISA was performed according to the manufacturer's protocol. For the determination of sICAM-1, three independent experiments in triplicate were carried out.

Statistical analysis

Results are reported as means with the standard error of the mean. Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc., version 4.01, 2004; San Diego, CA, USA). The group means were compared by one-way or two-way analysis of variance (ANOVA) with subsequent Bonferroni's multiple comparison test (dietary treatment against control) or Bonferroni post-tests comparing replicate means, respectively. Significance was set at P<.05.

Results

Plasma parameters

Feeding db/db mice for 14 days with RSG or HGTE resulted in reduced fasting plasma glucose concentrations compared to controls (Fig. 1). After 28 days, however, the blood glucose-lowering effect was only observed in the RSG group (Fig. 1, Table 3), whereas a reduced plasma sICAM concentration was found only in the HGTE group (Table 3). After 28 days, no differences were found in fasting plasma TG, NEFA, cholesterol, or insulin levels between the RSG or L/HGTE groups and controls (Table 3). In RSG-fed mice, lower fasting plasma glucose concentrations were paralleled by elevated plasma adiponectin concentrations compared to controls (Table 3). A two-way ANOVA indicated that effects on fasting plasma glucose concentrations are mainly attributable to the dietary treatment (32.44%, F=17.13, P<.0001) and the duration of dietary treatment (26.43%, F=41.71, P<.0001).

Table 3.

Fasting Plasma Parameters and Tissue Liver Weights of db/db Mice After 28-Day Treatment Period

Parameter	C	RSG	LGTE	HGTE
Glucose (mmol/L)	22.7±2.3	8.5±0.7^*	20.3±2.9	20.7±3.4
Δ_{14–28 day} Glucose (mmol/L)	11.4±2.1	1.1±0.9^*	9.7±2.8	13.1±3.5
TG (mmol/L)	1.5±0.1	1.5±0.1	1.8±0.2	1.8±0.2
NEFA (mmol/L)	0.8±0.05	0.7±0.09	0.8±0.09	0.9±0.1
CHOL (mmol/L)	4.9±0.4	4.6±0.4	4.7±0.2	5±0.2
Insulin (nmol/L)	1.8±0.2	2.2±0.3	1.5±0.2	1.2±0.1
ACRP30 (μg/mL)	6.1±0.3	12.0±0.9^*	5.8±0.3	6.2±0.2
sICAM-1 (ng/mL)	398±14	343±24	343±20	327±19^*
Liver^a (mg/g)	58.8±2.9	53.6±3.7	56±3.1	47.5±2.5^*

Reported as mg liver tissue/g body weight.

Data are means±standard error of the mean (n=8), one-way analysis of variance, ^* P<.05 compared to controls.

ACRP30, full-weight adiponectin; C, control; CHOL, total cholesterol; HGTE, high-dose green tea extract (1 g/kg diet); LGTE, low-dose green tea extract (0.1 g/kg diet); NEFA, nonesterified fatty acids; RSG, rosiglitazone (0.02 g/kg diet); sICAM, soluble intercellular adhesion molecule-1; TG, triacylglycerols.

Body weight and liver and periuterine fat mass

All animals continuously gained body mass during the feeding period (Fig. 2). However, at the end of the feeding period (days 21 and 28), the average body weight was higher in the RSG-fed group compared to controls (Fig. 2A). Conversely, body weights of L/HGTE-treated db/db mice did not differ compared to controls (Fig. 2). The mean liver wet weight was lowered by HGTE feeding (Table 3), while periuterine fat mass was reduced by ∼25% by RSG treatment compared to controls, despite these mice having the highest body weights (Fig. 2A, B).

FIG. 2.

Body weight development of db/db mice over 28 days (A) and periuterine fat mass after 28-day (B) feeding period without (C) or with the addition of RSG (0.02 g/kg diet) or green tea extract (0.1 and 1 g/kg diet, LGTE and HGTE). Data are means±SEM (n=8), (A) two-way ANOVA, (B) one-way ANOVA. *P<.05 compared to controls.

sICAM-1 release in stimulated EAhy926 endothelial-like cells

No sICAM-1 was detected in the supernatants of unstimulated EAhy926 endothelial cells. However, stimulation of EAhy926 cells with TNF-α markedly increased the release of sICAM-1. While RSG had no effect on sICAM-1 release in stimulated EAhy926 cells, GTE and EGCG reduced the release of sICAM-1 in stimulated EAhy926 cells (Fig. 3).

FIG. 3.

Effect of green tea extract on soluble intercellular adhesion molecule 1 (sICAM-1) release in tumor necrosis factor alpha (TNF-α)-stimulated EAhy926 cells. EAhy926 cells were preincubated (24 h) with dimethylsulfoxide (DMSO, 0.1%) as a negative control, RSG (1 μmol/L), green tea extract (GTE, 10 μg/mL), or epigallocatechin-3-gallate (EGCG, 25 μmol/L) after 24-h stimulation with TNF-α (1 ng/mL). Data are means+SEM (n=9), one-way ANOVA. **P<.01, ***P<.001 compared to stimulated control (stim).

Discussion

Although beneficial health effects on obesity, NIDDM, and/or coronary heart diseases due to the consumption of flavanol-rich green tea or oral EGCG supplementation have been suggested,^10,20
–22 the underlying mechanisms remain largely elusive. Thus, we investigated the glucose-lowering, antiadipogenic, and anti-inflammatory effects of dietary GTE in an animal model used extensively for the screening of antidiabetic agents.²³ The obese db/db mouse reliably simulates the development of insulin resistance, demonstrating progressive beta-cell failure with pronounced hyperglycemia, adiposity, and hypoadiponectinemia similar to that found in human NIDDM. In the present study, diet was supplemented with GTE (containing 34% EGCG; 0.1 or 1 g GTE/kg diet). Corresponding daily intake was ∼6 mg EGCG/kg body weight (LGTE) or ∼60 mg EGCG/kg body weight (HGTE), respectively. The lower dose was chosen to simulate an EGCG intake achievable by fortified daily green tea (nutrient data bank [NDB] No. 99070) consumption (0.5 L),²⁴ but was explicitly nonpharmacological. After 14 days of dietary intervention, body weights of mice did not differ. Groups supplemented with RSG or the high dose of GTE had improved fasting glycemia compared to controls, while fasting plasma glucose concentrations were not reduced in mice fed the low GTE dose. Thus, it seems that even in the early stages of insulin resistance, low doses of GTE do not exhibit glucose-lowering effects in female db/db mice. Continuous feeding of the respective diets resulted in higher body mass gain in mice fed RSG compared to controls, while mice fed GTE did not differ from controls. However, as insulin resistance advances, improved fasting glycemia, paralleled by unreduced plasma adiponectin, was only found in mice fed RSG after the 28-day feeding period. Neither of the two dietary GTE doses exhibited glucose-lowering effects in female db/db mice after 28 days. Thus, we assume that administration of nonpharmacological EGCG doses only transiently lowers plasma glucose as a consequence of progressive pancreatic β-cell failure in this animal model of obesity and diabetes. To date, neither epidemiological nor interventional studies provide clear-cut evidence for glucose-lowering effects of EGCG consumption in humans.^25

–28 Also, antiadipogenic effects of EGCG, reported in previous in vitro investigations,^15.29
–31 were not observed in this study. In the present study, animals were housed in groups of four with free access to food and water, and thus individual food intake was not assessed. Additionally, animals were fed a powdery diet resulting in extensive food wastage, and thus accurate measurement of a group-based feed consumption was not possible. However, the impact of RSG on feed intake and body weight gain has been investigated before. Despite reduced feed intake, RSG intake was paralleled by an increase in body weight, a known critical side effect of RSG intake.³² Nevertheless, the impact of EGCG on body weight remains controversial. Intraperitoneal injection of EGCG has been found to have dose-dependent antiobesity effects in Sprague-Dawley rats and in lean and obese Zucker rats.³³ In the same study, oral EGCG supplementation reduced food intake by 50%–60%, causing a resultant reduction in weight gain.³³ Oral EGCG supplementation (10 g/kg diet) prevented diet-induced increases in body weight in C57BL/6J mice treated for 5 months, and reversed established obesity in Sprague-Dawley rats treated for 4 weeks.¹⁵ However, 10 g EGCG/kg diet did not reduce weight gain in db/db mice unless fed for 9 weeks. Thus, it can be concluded that antiadipogenic effects of EGCG are mainly dose-, but also time and model dependent, and are not established for EGCG intake achievable by even fortified daily green tea consumption.

Hypoadiponectinemia is associated with adiposity and impaired insulin sensitivity.³⁴ Adiponectin synthesis is stimulated upon ligand binding to the transcription factor PPAR-γ, which is the major target of thiazolidinones.³ In the present study, hypoadiponectinemia was prevented, irrespective of higher weight gain, only in the RSG-supplemented group, suggesting that GTE has no PPAR-γ ligand activity in db/db mice at the doses employed herein.

Inflammation is a feature common in both obesity and insulin resistance, often accompanied by elevated cytokine secretion from adipose tissue. In the literature, anti-inflammatory effects of RSG are discussed controversially. Cytokines, such as TNF-α and nuclear factor kappa-light-chain enhancer of activated B-cell (NF-κB), induce the expression and release of adhesion molecules (ICAM-1) in different cell types.³⁵ In chronic inflammatory diseases, sICAM-1 occurs in the circulation and can be used as a biomarker of inflammation.³⁶ On one hand, RSG treatment has been shown to reduce cytokine expression,³⁷ but on the other hand, it fails to reduce TNF-α mRNA concentrations in white adipose tissue of db/db mice.^38,39 EGCG, however, has previously been reported to inhibit TNF-α secretion and thus NF-κB function in several cell lines.^40
–42 In the present study, only GTE and EGCG reduced sICAM-1 release in stimulated EAhy926 cells, and only high-dose GTE reduced sICAM release in db/db mice. This might contribute to a hepatoprotective effect. During chronic low-grade inflammation, numerous mediators are released from immune cells and adipocytes that contribute to liver damage and liver disease, associated with hepatic steatosis, necroinflammation, apoptosis, and fibrosis.⁴³ Despite having no improvement in plasma triacylgylcerol, NEFA, and cholesterol concentrations, mice fed HGTE displayed decreased liver wet weights compared to controls. Although we have not measured hepatic TG content in mice, liver wet weight together with reduced plasma sICAM concentrations is be suggestive of some hepatoprotective effects of GTE.

In summary, oral green tea extract supplementation at nonpharmacological EGCG doses had no adiponectin-inducing or antiadipogenic effects in db/db mice. However, a higher dose of green tea extract, and therefore EGCG, transiently improved glycemia and effectively reduced sICAM-1 release, and thus might exert anti-inflammatory and hepatoprotective effects in the progressive diabetic state.

Footnotes

Acknowledgment

We are grateful to Wiebke Kühl (Institute of Animal Nutrition & Physiology, Christian Albrechts University of Kiel, Germany) for excellent technical assistance.

Author Disclosure Statement

No competing financial interests exist

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