Caffeic acid phenethyl amide improves glucose homeostasis and attenuates the progression of vascular dysfunction in Streptozotocin-induced diabetic rats

Abstract

Background: Glucose intolerance and cardiovascular complications are major symptoms in patients with diabetes. Many therapies have proven beneficial in treating diabetes in animals by protecting the cardiovascular system and increasing glucose utilization. In this study, we evaluated the effects of caffeic acid phenethyl amide (CAPA) on glucose homeostasis and vascular function in streptozotocin (STZ)-induced type 1 diabetic rats.

Methods: Diabetes (blood glucose levels > 350 mg/dL), was induced in Wistar rats by a single intravenous injection of 60 mg/kg STZ. Hypoglycemic effects were then assessed in normal and type 1 diabetic rats. In addition, coronary blood flow in Langendorff-perfused hearts was evaluated in the presence or absence of nitric oxide synthase (NOS) inhibitor. The thoracic aorta was used to measure vascular response to phenylephrine. Finally, the effect of chronic treatment of CAPA and insulin on coronary artery flow and vascular response to phenylephrine were analyzed in diabetic rats.

Results: Oral administration of 0.1 mg/kg CAPA decreased plasma glucose in normal (32.9 ± 2.3% decrease, P < 0.05) and diabetic rats (11.8 ± 5.5% decrease, P < 0.05). In normal and diabetic rat hearts, 1-10 μM CAPA increased coronary flow rate, and this increase was abolished by 10 μM NOS inhibitor. In the thoracic aorta, the concentration/response curve of phenylephrine was right-shifted by administration of 100 μM CAPA. Coronary flow rate was reduced to 7.2 ± 0.2 mL/min at 8 weeks after STZ-induction. However, 4 weeks of treatment with CAPA (3 mg/kg, intraperitoneal, twice daily) started at 4 weeks after STZ induction increased flow rate to 11.2 ± 0.5 mL/min (P < 0.05). In addition, the contractile response induced by 1 μM phenylephrine increased from 6.8 ± 0.6 mN to 11.4 ± 0.4 mN (P < 0.05) and 14.9 ± 1.4 mN (P < 0.05) by insulin (1 IU/kg, intraperitoneal) or CAPA treatment, respectively.

Conclusions: CAPA induced hypoglycemic activity, increased coronary blood flow and vascular response to phenylephrine in type 1 diabetic rats. The increase in coronary blood flow may result from endothelial NOS activation. However, the detailed cellular mechanisms need to be further evaluated.

Figure 1

The structures of CAPE and CAPA, and the synthetic process of CAPA. CAPA was obtained from the amide binding coupling method, beginning with caffeic acid. CAPA: R=−(CH₂)₂Ph. benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), dichloromethane (CH₂Cl₂), triethylamine (Et3N), dimethylformamide (DMF).

Figure 2

The effect of CAPA on the glucose tolerance test.Intravenous glucose tolerance tests (IVGTT) were performed in anesthetized fasted animals. Rats were orally administered CAPA (0.5 mg/mL/kg, n = 6) or the same volume of distilled water (vehicle, n = 8) at 30 minutes before intravenous injection of glucose (1000 mg/kg). Blood glucose levels were measured before and at 5, 10, 20, 40, 60, 90, and 120 min after glucose injection. (●) CAPA-treated group: CAPA (0.5 mg/mL/kg, p.o. 30 min before glucose intravenous injection). (○) Vehicle-treated group: Rats treated with vehicle at the same volume were used as controls. Data are presented as the mean ± SEM. *P < 0.05 compared with the vehicle.

Figure 3

Effect of CAPA on coronary arterial flow rate in normal or l-NAME-treated rat hearts. Coronary arterial flow rate (mL/min) was measured after 30 min of equilibrium with retrograde perfusion using a constant pressure of 80 mmHg. The l-NAME-treated hearts were perfused with 10 μM l-NAME (A), and data were normalized to basal flow rate (B). Data (mean ± SEM) were obtained from 4–9 animals. *P < 0.05 compared with the untreated control of normal rat hearts and ^#P < 0.05 compared with the untreated control of the L-NAME group.

Figure 4

Concentration-dependent inhibition of CAPA on thoracic aorta, constriction-induced by high concentration potassium or phenylephrine. The relaxation effect of CAPA was measured in endothelium-intact (+EC) and endothelium-denuded (−EC) thoracic aorta. The aortic strips were pre-constricted with high concentration of potassium (80 mM) (A) or 1 μM phenylephrine (B), pre-treated with inhibitors (C), such as 10 μM NOS inhibitor (l-NAME), 10 μM nitric oxide-sensitive guanylyl cyclase selective inhibitor (ODQ), and 100 μM soluble guanylyl cyclase inhibitor (MB), and CAPA was added in a cumulated concentrations in the organ bath to assess relaxation effects. The IC₅₀ values for CAPA are shown in (D). Data (mean ± SEM) were obtained from 6–8 animals.

Figure 5

Effect of CAPA on concentration-dependent contractile response to phenylephrine in endothelium-denuded aortic strips. The effect of CAPA on the contractile response to phenylephrine was measured in endothelium-denuded thoracic aorta. The aortic strips were pre-treated with 1, 10 and 100 μM CAPA, and phenylephrine was added in a cumulative dose in the organ bath (A). Normalized contraction to the maximum response of phenylephrine is shown (B). The EC₅₀ values for phenylephrine and the Hill coefficient are shown in (C) and (D). Data (mean ± SEM) were obtained from 6–8 animals. *P < 0.05 compared with the vehicle, DMSO pre-treatment group.

Figure 6

Effect of chronic treatment with CAPA and insulin on the vascular response. (A) The basal coronary arterial flow rate (mL/min) in retrograde-perfused hearts was measured to evaluate the effect of CAPA in type 1 diabetic rats. The animals were divided into 4 groups and intraperitoneally administered CAPA twice daily for 4 weeks. Control: age- and sex-matched normal rats, n = 6. STZ-veh: vehicle-treated diabetic rats, 0.1 mL/kg DMSO, n = 4. STZ-insulin: insulin-treated diabetic rats, 1 IU/kg, n = 4. STZ-CAPA: CAPA-treated diabetic rats, 3 mg/kg, n = 5. ^*,#P < 0.05 compared with control groups and STZ-induced diabetic rats treated with vehicle. (B) Effect of CAPA on the phenylephrine-induced aortic constriction of type 1 diabetic rat aorta. The effect of CAPA on the phenylephrine-induced aortic contraction response (mN) of STZ-induced type 1 diabetic rat thoracic aorta was measured in isolated aortic strips. The animals were divided into 4 groups. Control: age- and sex-matched normal rats, n = 4. STZ-veh: vehicle-treated diabetic rats, 0.1 mL/kg DMSO, n = 4. STZ-Ins: insulin-treated diabetic rats, 1 IU/kg, n = 4. STZ-CAPA: CAPA-treated diabetic rats, 3 mg/kg, n = 6. ^*,#P < 0.05 compared with control and STZ-veh groups.