Fig. 1Effect 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. 2Effect 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. 3Effect 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. 4Effect 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. 5Effect 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. 6Effect 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.
Table 1Food intake and hypothalamic expression of neuropeptide Y (NPY; C) mRNA after the drug treatments