Effect of Green Tea Extract/Poly-γ-Glutamic Acid Complex in Obese Type 2 Diabetic Mice

Abstract

Background: The increasing prevalence of type 2 diabetes mellitus (T2DM) is associated with the rapid spread of obesity. Obesity induces insulin resistance, resulting in β-cell dysfunction and thus T2DM. Green tea extract (GTE) has been known to prevent obesity and T2DM, but this effect is still being debated. Our previous results suggested that circulating green tea gallated catechins (GCs) hinders postprandial blood glucose lowering, regardless of reducing glucose and cholesterol absorption when GCs are present in the intestinal lumen. This study aimed to compare the effect of GTE with that of GTE coadministered with poly-γ-glutamic acid (γ-PGA), which is likely to inhibit the intestinal absorption of GCs.

Methods: The db/db mice and age-matched nondiabetic mice were provided with normal chow diet containing GTE (1%), γ-PGA (0.1%), or GTE+γ-PGA (1%:0.1%) for 4 weeks.

Results: In nondiabetic mice, none of the drugs showed any effects after 4 weeks. In db/db mice, however, weight gain and body fat gain were significantly reduced in the GTE+γ-PGA group compared to nondrug-treated db/db control mice without the corresponding changes in food intake and appetite. Glucose intolerance was also ameliorated in the GTE+γ-PGA group. Histopathological analyses showed that GTE+γ-PGA-treated db/db mice had a significantly reduced incidence of fatty liver and decreased pancreatic islet size. Neither GTE nor γ-PGA treatment showed any significant results.

Conclusion: These results suggest that GTE+γ-PGA treatment than GTE or γ-PGA alone may be a useful tool for preventing both obesity and obesity-induced T2DM.

Keywords: Diabetes mellitus, type 2; Gallated catechins; Glucose intolerance; Obesity; Poly-γ-glutamic acid; db/db mice.

Fig. 1

Effect of green tea extract (GTE)+poly-γ-glutamic acid (γ-PGA) on oral glucose loading in normal mice. Normal mice were fasted for 12 hours and then given water, GTE or GTE+γ-PGA orally in the concentrations indicated in Methods. (A) Ninety minutes later, glucose (2 g/kg) was administered orally. A blood sample was collected from the tail vein at the indicated time. (B) The area under the curve (AUC) is depicted as the percentage of the control value. ANOVA with Bonferroni correction. (C) Assumed intermolecular interactions between epigallocatechin gallate (EGCG) and γ-PGA. In the diagram, -O-H---O- (hydrogen bonding [HB], blue), -O-H---O=C- (HB, violet), -NH---O(H)- (HB, green), and C (aromatic)---CH- (van der Waals interaction [VW], red) interactions are shown. ^aP<0.05 compared to the control value; n=7 for each group.

Fig. 2

Effect of green tea extract (GTE)+poly-γ-glutamic acid (γ-PGA) on body weight (BW) and body fat after 4 weeks of treatment. From 10 weeks of age, db/db mice and age-matched control nondiabetic heterozygous mice were provided with a semisynthetic, a normal chow diet containing γ-PGA, GTE or GTE+γ-PGA. After 4 weeks, BW (A, B) and weight of visceral adipose tissues (C, D) were measured. ANOVA with Bonferroni correction. WAT, white adipose tissue. ^aP<0.05 compared to the db/db control value; n=10 for each group.

Fig. 3

Effect of green tea extract (GTE)+poly-γ-glutamic acid (γ-PGA) on fasting blood glucose, fasting plasma insulin and homeostasis model assessment of insulin resistance (HOMA-IR) index after 4 weeks of treatment. After 4 weeks, the mice were fasted for 12 hours. (A, B) Blood samples were collected from the tail vein, and fasting blood glucose and plasma insulin levels were measured. (C) HOMA-IR was calculated using the fasting blood glucose and insulin levels. ANOVA with Bonferroni correction. ^aP<0.05 compared to the db/db control value; n=10 for each group.

Fig. 4

Effect of green tea extract (GTE)+poly-γ-glutamic acid (γ-PGA) on glucose tolerance after 4 weeks of treatment. Changes in blood glucose levels during intraperitoneal glucose tolerance test, which was performed after 4 weeks of treatment with diet containing γ-PGA, GTE, or GTE+γ-PGA. Mice were fasted for 12 hours, and then 500 mg/kg glucose was injected in nondiabetic control (A) and db/db (B) mice. The area under the curve (AUC) was depicted in (C) as the percentage of the db/db control value. ANOVA with Bonferroni correction. ^aP<0.05 compared to the db/db control value; n=10 for each group.

Fig. 5

Effect of green tea extract (GTE)+poly-γ-glutamic acid (γ-PGA) on pancreatic islet size after 4 weeks of treatment. At the conclusion of the study, the mice were anaesthetized, and the pancreas was dissected out. The pancreas was fixed immediately in 10% neutral formalin solution and embedded in paraffin. (A) Embedded pancreas tissue blocks were cut into 6-micron sections and stained with horseradish peroxidase conjugated anti-insulin antibody (original magnification, ×20). (B) Islet size was measured by the size of the stained area under the same magnification. ANOVA with Bonferroni correction. ^aP<0.05 compared to the db/db control value; n=10 for each group.

Fig. 6

Effect of green tea extract (GTE)+poly-γ-glutamic acid (γ-PGA) on fatty liver change and hepatic triglyceride (TG) content after 4 weeks of treatment. At the conclusion of the study, the mice were anaesthetized, and the liver was dissected. The liver was fixed immediately in 10% neutral formalin solution and embedded in paraffin. (A) Embedded liver tissue blocks were cut into 6-micron sections and stained with hematoxylin and eosin (original magnification, ×20). (B) Hepatic TG content (mg/g protein) was measured by enzyme-linked immunosorbent assay. ANOVA with Bonferroni correction. ^aP<0.05 compared to the db/db control value; n=10 for each group.