EGCG-derived polymeric oxidation products enhance insulin sensitivity in db/db mice

Abstract

The present study investigated the influence of epigallocatechin-3-gallate (EGCG) and its autoxidation products on insulin sensitivity in db/db mice. Compared to EGCG, autoxidation products of EGCG alleviated diabetic symptoms by suppressing the deleterious renal axis of the renin-angiotensin system (RAS), activating the beneficial hepatic axis of RAS, and downregulating hepatic and renal SELENOP and TXNIP. A molecular weight fraction study demonstrated that polymeric oxidation products were of essential importance. The mechanism of action involved coating polymeric oxidation products on the cell surface to protect against cholesterol loading, which induces abnormal RAS. Moreover, polymeric oxidation products could regulate RAS and SELENOP at doses that were far below cytotoxicity. The proof-of-principal demonstrations of EGCG-derived polymeric oxidation products open a new avenue for discovering highly active polymeric oxidation products based on the oxidation of naturally occurring polyphenols to manage diabetes and other diseases involving abnormal RAS.

Keywords: EGCG autoxidation Products; Insulin sensitivity; Renin-angiotensin system; SELENOP; TXNIP; Type 2 diabetes.

Graphical abstract

Fig. 1

In vitro characterization and in vivo hypoglycemic effect of EGCG and EAOP. (A–D)In vitro characterization. (A) color profile; (B) molecular weight distribution; (C) scavenging ROS in selenite/glutathione system; (D) scavenging hydroxyl radical in copper/glutathione system. Data are presented as the mean ± range (n = 2). (E–H)In vivo hypoglycemic effect. db/db mice (n = 6/group) were i.p. administered with E0 (EGCG), E16, and E64, respectively, at a dose of 10 mg/kg daily for three weeks. (E) Fasting blood glucose. (F) Urine output. (G) Water intake. (H) Food intake. (I) Body weight. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control.

Fig. 2

Effects of E64 in db/db mice. db/db mice (n = 6/group) were i.p. administered with E64 at a dose of 5 or 10 mg/kg daily (referred to as E64-5 and E64-10, respectively) for three weeks. (A) Fasting blood glucose. (B) Plasma insulin. (C) HOMA-IR. (D) ITT. (E) ITT-AUC. (F) Urine output. (G) Water intake. (H) Food intake. (I) Body weight. Data are presented as the mean ± SEM. **P < 0.01 and ***P < 0.001, compared to the control.

Fig. 3

Influence of E64 on hepatic and renal RAS. db/db mice (n = 6/group) were i.p. administered with E64 a dose of 5 or 10 mg/kg daily (referred to as E64-5 and E64-10, respectively) for three weeks. (A) Hepatic RAS. (B) Hepatic ACE2 activity. (C) Renal RAS. (D) Renal ACE2 activity. Data are presented as the mean ± SEM. **P < 0.01 and ***P < 0.001, compared to the control.

Fig. 4

E64 regulates multiple targets associated with insulin sensitivity. db/db mice (n = 6/group) were i.p. administered with E64 at a dose of 5 or 10 mg/kg daily (referred to as E64-5 and E64-10, respectively) for three weeks. (A) Hepatic SELENOP and TXNIP proteins. (B) Hepatic GPx activity. (C) Hepatic Trx activity. (D) Renal SELENOP and TXNIP proteins. (E) Renal Trx activity. (F) Serum SELENOP. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control.

Fig. 5

In vitro characterization and in vivo hypoglycemic effect of different molecular weight fractions of E64. (A–C)In vitro characterization: (A) Color profile. (B) Scavenging ROS in selenite/glutathione system. (C) Scavenging hydroxyl radical in copper/glutathione system. Data are presented as the mean ± range (n = 2). (D–I)In vivo hypoglycemic effect: db/db mice (n = 6/group) were i.p. administered with the three fractions of E64 (<10 kDa, 10–50 kDa, and >50 kDa) at a dose of 5 mg/kg daily for four weeks. (D) Urine output. (E) Water intake. (F) Food intake. (G) Body weight. (H) Fasting blood glucose. (I) Plasma insulin level. (J) HOMA-IR. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control.

Fig. 6

Influence of the >50 kDa fraction on RAS, SELENOP, and TXNIP. db/db mice (n = 6/group) were i.p. administered with the >50 kDa fraction at a dose of 5 mg/kg daily for four weeks. (A) Skeletal muscular RAS. (B) Hepatic RAS. (C) Renal RAS. (D) Abdominal adipose RAS. (E) Pancreatic RAS. (F) Pancreatic SELENOP and TXNIP. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control.

Fig. 7

Influence of the >50 kDa fraction on gluconeogenesis and GLUT4. db/db mice (n = 6/group) were i.p. administered with the >50 kDa fraction at a dose of 5 mg/kg daily for four weeks. (A) Renal G6Pase-α and PEPCK. (B) Hepatic G6Pase-α and PEPCK. (C) Skeletal, muscular GLUT4. (D) Abdominal adipose GLUT4. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control.

Fig. 8

Influence of the >50 kDa fraction on abnormal RAS induced by cholesterol in HK-2 cells. (A) Dose-dependent RAS regulation. Data are presented as the mean ± SEM (n = 3). **P < 0.01, compared to the control. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001, compared to cholesterol treatment. (B) Coating characterization. HK-2 cells were treated with the >50 kDa fraction (100 μg/mL) for 24 h, then the cells were lysed on ice for 3 min and centrifuged at 10,000 g for 5 min.