Diet supplementation with green tea extract epigallocatechin gallate prevents progression to glucose intolerance in db/db mice

Abstract

Background: Green tea was suggested as a therapeutic agent for the treatment of diabetes more than 70 years ago, but the mechanisms behind its antidiabetic effect remains elusive. In this work, we address this issue by feeding a green tea extract (TEAVIGO™) with a high content of epigallocatechin gallate (EGCG) or the thiazolidinedione PPAR-γ agonist rosiglitazone, as positive control, to db/db mice, an animal model for diabetes.

Methods: Young (7 week-old) db/db mice were randomized and assigned to receive diets supplemented with or without EGCG or rosiglitazone for 10 weeks. Fasting blood glucose, body weight and food intake was measured along the treatment. Glucose and insulin levels were determined during an oral glucose tolerance test after 10 weeks of treatment. Pancreata were sampled at the end of the study for blinded histomorphometric analysis. Islets were isolated and their mRNA expression analyzed by quantitative RT-PCR.

Results: The results show that, in db/db mice, EGCG improves glucose tolerance and increases glucose-stimulated insulin secretion. EGCG supplementation reduces the number of pathologically changed islets of Langerhans, increases the number and the size of islets, and heightens pancreatic endocrine area. These effects occurred in parallel with a reduction in islet endoplasmic reticulum stress markers, possibly linked to the antioxidative capacity of EGCG.

Conclusions: This study shows that the green tea extract EGCG markedly preserves islet structure and enhances glucose tolerance in genetically diabetic mice. Dietary supplementation with EGCG could potentially contribute to nutritional strategies for the prevention and treatment of type 2 diabetes.

Figure 1

Improved glycemic control in db/db mice after 10 weeks of treatment with EGCG or rosiglitazone. Mice received dietary supplementation with 1% (w/w) EGCG or 0.0021% (w/w) rosiglitazone (Rosi) for 10 weeks. Shown are fasting blood glucose levels (A), fasting plasma insulin levels (B), blood glucose concentrations (C, D) and plasma insulin concentrations (E, F) during an oral glucose tolerance test (OGTT). Raw data from OGTT are presented in C and E while calculated area under the curve is presented in D and F for blood glucose and plasma insulin respectively. Values represent mean ± SEM for 9 mice in each group. * denotes P < 0.05 for a chance difference vs controls using one-way ANOVA in conjunction with Dunnett's multiple comparison test.

Figure 2

Changes in insulin resistance after 10 weeks of treatment with EGCG or rosiglitazone. Peripheral insulin sensitivity was assessed in control db/db mice and db/db mice receiving a diet supplemented with 1% (w/w) EGCG or 0.0021% (w/w) rosiglitazone (Rosi) for 10 weeks. Insulin resistance was evaluated by two different means: Shown in A is homeostasis model assessment-estimated insulin resistance (HOMA-IR) that was calculated as fasting glucose (mM) * fasting insulin (μU/ml)/22.5. Quantitative Insulin Sensitivity check (QUICKI) is shown in B and was calculated as 1/[log ((fasting glucose (mg/dl) + (fasting insulin (μU/ml))]. Bars represent mean ± SEM for 9 mice in each group. * denotes P < 0.05 for a chance difference vs controls using one-way ANOVA in conjunction with Dunnett's multiple comparison test.

Figure 3

Effects of 10 weeks of treatment with EGCG or rosiglitazone on pancreatic islet morphology and β cell area. Light microscopic appearance and histomorphometric analysis of pancreatic islet morphology and β-cell area in control db/db mice and db/db mice receiving dietary supplementation with 1% (w/w) EGCG or 0.0021% (w/w) rosiglitazone (Rosi) for 10 weeks. H&E staining was used and original magnification was 200 x. Bars represent mean ± SEM for 9 mice in each group. * denotes P < 0.001 (number of pathological islets) and P < 0.01 (β cell area) for chance differences vs controls using one-way ANOVA in conjunction with Dunnett's multiple comparison test.

Figure 4

Increased number of pancreatic islets after 10 weeks of treatment with EGCG or rosiglitazone. Light microscopic appearance of pancreatic islets immunostained for insulin in control db/db mice and db/db mice receiving dietary supplementation with 1% (w/w) EGCG or 0.0021% (w/w) rosiglitazone (Rosi) for 10 weeks. Islets are immunostained for insulin (brown) and original magnification was 100 x. Bars represent mean ± SEM for 9 mice in each group. * denotes P < 0.05 for a chance difference vs controls using one-way ANOVA in conjunction with Dunnett's multiple comparison test.

Figure 5

Increased size and endocrine area of pancreatic islets after 10 weeks of treatment with EGCG or rosiglitazone. Light microscopic appearance of pancreatic islets immunostained for insulin in control db/db mice and db/db mice receiving dietary supplementation with 1% (w/w) EGCG or 0.0021% (w/w) rosiglitazone (Rosi) for 10 weeks. Islets are immunostained for insulin (brown) and original magnification was 200 x. Bars represent mean ± SEM for 9 mice in each group. For islet size, * denotes P < 0.001, and for endocrine area P < 0.01, for chance differences vs controls using one-way ANOVA in conjunction with Dunnett's multiple comparison test.

Figure 6

EGCG treatment of isolated islets of Langerhans ex vivo fails to prevent palmitate-induced phosphorylation of JNK and formation of cleaved caspase 3. Pancreatic islets were isolated from lean C57Bl/6J mice and exposed ex vivo for 24 hours in the absence or presence of 0.5 mM palmitate with or without 5, 10 or 20 μM EGCG. Western blot analyses for phosphorylation of JNK1 (A), JNK2 (B) and formation of the apoptosis marker cleaved caspase 3 (C). Bars represent mean ± SEM for 5 different islet preparations in each group. * denotes P < 0.05 for a palmitate effect, and # denotes P < 0.05 for an EGCG effect using one-way ANOVA in conjunction with Dunnett's multiple comparison test. Representative blots for phosphorylated JNK1 (p-JNK1), phosphorylated JNK2 (p-JNK2) and cleaved caspase 3 (CC3) are shown in D.

Figure 7

EGCG in vitro partially protects MIN6 cells from palmitate-induced apoptosis but does not influence palmitate-induced phosphorylation of JNK. MIN6 cells were treated with 0.5 mM palmitate in the absence or presence of 20 μM EGCG for 48 hours. Western blot analysis of formation of the apoptosis marker cleaved caspase 3 (A), phosphorylation of JNK1 (C) and JNK2 (D). DNA fragmentation (B) was measured with an ELISA against cytoplasmic oligonucleosomes. Bars represent mean ± SEM for 4 different cell preparations in each group. * denotes P < 0.05 for a palmitate effect, and # denotes P < 0.05 for an EGCG effect using one-way ANOVA in conjunction with Dunnett's multiple comparison test. Representative blots for phosphorylated JNK1 (p-JNK1), phosphorylated JNK2 (p-JNK2) and cleaved caspase 3 (CC3) are shown in E.