Rosmarinic acid improves hypertension and skeletal muscle glucose transport in angiotensin II-treated rats

Rosmarinic acid was purchased from Sigma–Aldrich Inc. (St. Louis, MO). Angiotensin II was purchased from AnaSpec Inc. (Fremont, CA). Rat insulin radioimmunoassay (RIA) kits were purchased from Millipore (St. Charles, MO). Glucose enzymatic colorimetric tests were purchased from HUMAN Gesellschaft fÜr Biochemica und Diagnostica mbH (Wiesbaden, Germany). 2-[1,2-³H] deoxyglucose and [U-¹⁴C] mannitol were purchased from PerkinElmer Life Sciences (Boston, MA). Antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA).

Experiments were carried out using 8-week-old male Sprague Dawley rats weighing 260–290 g from the National Laboratory Animal Center, Nakhon Pathom, Thailand. All rats were housed in a strict hygienic conventional housing system. Each rat was placed in a 9 × 12 × 6 in. cage with corn cob bedding at the Center of Animal Facilities, Faculty of Science, Mahidol University. The room temperature was controlled at 22 °C with a 12:12-h light-dark cycle (light on from 0600 to 1800 h). Rats had free access to water and pellet rat chow (Perfect Companion, Samutprakarn, Thailand). One week after arrival, rats were randomly assigned into the SHAM (control groups, n = 10 rats/group) and ANG II-treated groups (experimental groups, n = 10 rats/group). The sample size was calculated from blood pressure data according to Karthik et al., 2011 [2] by using Minitab 14 (Minitab Inc., State College, PA). ANG II (250 ng/kg/min) was subcutaneously delivered for 14 days by implanting a mini-osmotic pump (model 2002, DURECT Corporation, Cupertino, CA) on the back and slightly posterior to the scapulae. To study the acute effects of RA, 14-day ANG II-treated rats received a single dose of 10, 20, or 40 mg/kg RA by a single gavage. A pharmacokinetic study of RA had reported that the t_1/2 of RA was 63.9 min [11]. The distribution of RA in skeletal muscle tissue had been observed 30 min after a single gavage [12]. Therefore, blood and tissue were collected 30 min after a single gavage, and the concentration of RA in blood and tissues was expected to be high. To assess the chronic effects of RA and to minimize the acute effects of RA, blood and tissues were collected at least 16 h after the most recent treatment. This study design was previously used in our study to evaluate the chronic effects of Curcuma comosa Roxb. on whole-body and skeletal muscle insulin sensitivity [13]. Rats in the SHAM and ANG II groups were gavaged with water and considered controls. In a separate study, the chronic effects of RA were assessed in rats that received 10, 20, or 40 mg/kg RA by gavage at 1600–1700 h for 14 consecutive days. Blood pressure was measured weekly by a tail cuff plethysmography apparatus using the Coda Monitoring system (Kent Scientific Corporation, Torrington, CT). Blood and tissue collections were performed at 0900–1200 h. Before tissue collection, rats were deeply anesthetized by intraperitoneal injection of thiopental (100 mg/kg). Respiratory rate, responses to noxious stimuli, and spontaneous responses were observed throughout the collection. After muscle dissection, other tissues were collected, and the rats were sacrificed by removal of the heart.

Glucose tolerance tests were performed to determine the whole-body insulin sensitivity. In the evening (1800 h) on the day before the test, rats were restricted to 4 g of chow. In the next morning (0800–0900 h), rats were gavaged one time with 1 g/kg of glucose. Tail blood was collected into microfuge tubes containing anticoagulant (18 mM final concentration of EDTA) before and 15, 30, 60, and 120 min after the glucose feeding (1 g/kg). The blood samples were centrifuged at 13000×g at 4 °C for 1 min. Then, plasma samples were collected to determine glucose and insulin concentrations [14]. After the test, each rat was given sterile 0.9% saline subcutaneously as soon as possible for the replacement of the body fluid loss. Furthermore, plasma insulin and glucose concentrations were measured by RIA and enzymatic colorimetric tests, respectively.

Forty-eight hr after performing the OGTT, rats were restricted to 4 g of chow at 1800 h. Each rat was weighed and deeply anesthetized with an intraperitoneal injection of thiopental (100 mg/kg) before a dissection of soleus muscle. Then, soleus muscle was subsequently divided into two strips. Each muscle strip (~ 25 mg) was incubated at 37 °C for 60 min in 3 ml of oxygenated Krebs–Henseleit buffer (KHB) supplemented with 8 mM D-glucose, 32 mM D-mannitol, 0.1% radioimmunoassay-grade bovine serum albumin, and the presence or absence of 2 mU/ml insulin. After incubation, the muscle strips were rinsed at 37 °C for 10 min in 3 ml of oxygenated Krebs–Henseleit buffer (KHB) containing 40 mM mannitol and insulin, if previously present. Finally, the muscle strips were incubated for 20 min in 2 mL of KHB containing 1 mmol/L 2-[1,2-³H] deoxyglucose (2-DG (300 μCi/mmol), 39 mmol/L [U-¹⁴C] mannitol (0.8 μCi/mmol), 0.1% BSA, and insulin, if previously present. Each flask was gassed with 95% O₂–5% CO₂ throughout the incubation period of the experiment. At the end of the incubation, the muscle strips were removed from the flasks, had the excess fat and connective tissue trimmed off, were frozen with liquid nitrogen, and immediately weighed. Then, the muscle strips were solubilized in 0.5 ml of 0.5 N NaOH for 1 h and mixed with 10 ml of a scintillation cocktail. The specific intracellular accumulation of 2–DG was determined by subtracting the ³H activity in the extracellular space from the total ³H activity in each muscle strip [15]. The specific intracellular accumulation of 2–DG was determined using mannitol to correct for the extracellular accumulation of 2–DG. Glucose transport activities were measured as the intracellular accumulation of 2–DG (in pmol/mg muscle wet weight/20 min) [15].

The soleus muscle from the other leg was dissected and subsequently divided into two strips. The muscle strips were incubated in the same solution type that was used for measuring GT in the presence or absence of 2 mU/ml insulin. After incubation, each muscle strip was trimmed of excess fat and connective tissue, quickly frozen in liquid nitrogen and kept at − 80 °C until performing immunoblotting. The muscle strips were homogenized in ice-cold lysis buffer: 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 2 mM EDTA, 10 mM NaF, 20 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 10% glycerol, 1% Triton X-100, 2 mM Na₃VO₄, 10 μg/ml aprotinin and leupeptin, and 2 mM PMSF. After a 20-min incubation on ice, the homogenates were centrifuged at 13000×g for 20 min at 4 °C. Proteins in the homogenate were separated on polyacrylamide gel and transferred electrophoretically onto nitrocellulose paper. The blots were incubated with an appropriate dilution of commercially available antibodies (Cell Signaling Technology Inc., Beverly, MA) against phospho-Akt (Ser473) (#9271; 1:800), Akt (#9272; 1:800), phospho-GSK-3α/β (Ser21/9) (#9331S; 1:1000), GSK-3α/β (#5676S; 1:1000), phospho-ERK1/2 (Thr202/Tyr204) (#4377; 1:1000), ERK1/2 (#4695; 1:1000), phospho-p38 MAPK (Thr180/Tyr182) (#9211; 1:800), p38 MAPK (#9212; 1:800), phospho-SAPK/JNK (Thr183/Tyr185) (#9251; 1:800), SAPK/JNK (#9252; 1:1000), and GAPDH (#2188; 1:3000). Subsequently, all blots were incubated with anti-rabbit IgG HRP-linked antibody (#7074; 1:1500). Protein bands were visualized by enhanced chemiluminescence. Images were digitized on a C-Digit Blot Scanner (LI- COR Biotechnology, Lincoln, NE), and band intensities were quantified using Image Studio Software version 3.1.

After administration of ANG II for 14 days, systolic, diastolic, and mean arterial blood pressure increased approximately 30–40 mmHg relative to the first week after ANG II administration. At the end of the study, ANG II increased blood pressure levels by 49–63 mmHg (Fig. 1, P < 0.05). The final body weights of the ANG II rats were significantly reduced compared with the SHAM rats (Table 1 and Table 2). At the end of the experiment, the liver weight to body weight ratio was not significantly changed, whereas the heart weight to body weight ratio increased by 0.77–0.95 g/kg (Table 1 and Table 2; P < 0.05).

Chronic infusion of ANG II increased fasting plasma glucose (1.29 and 1.54 mmol/l) and decreased insulin AUC (1.62 and 2.00 μU/ml/min*10³) levels when compared to SHAM conditions (Table 1 and Table 2; P < 0.05). However, there was no significant change in whole-body insulin sensitivity, including the homeostasis model assessment-estimated insulin resistance (HOMA-IR) and the glucose-insulin (G-I) index. Meanwhile, the study did not find any significant change from the ANG II infusion in slow-twitch muscle glucose transport activities (Fig. 2) and its protein elements (Fig. 3).

All doses of acute and chronic RA treatment attenuated the blood pressure-increasing effects of ANG II. A reduction in blood pressure was found for all doses of acute RA treatment with means decreased by 46–64 mmHg, and for all chronic RA treatments, with means decreased by 33–58 mmHg (Fig. 1; P < 0.05). As shown in Table 1 and Table 2, liver weight to body weight ratios were not altered after RA treatment. Acute treatment with RA and chronic treatment with 10 mg/kg RA resulted in significantly increased heart weight to body weight ratios as was observed in the ANG II groups.

The fasting plasma glucose in the ANG II-treated rats was reduced by 1.17 mmol/l after a single gavage of 40 mg/kg RA. On the other hand, the fasting plasma glucose were decreased in the chronic RA treatment groups (10, 20, and 40 mg/kg) by 0.94–1.04 μU/ml/min*10³ (Table 1 and Table 2; P < 0.05). Neither acute nor chronic treatment with RA altered the HOMA-IR or G-I index. Interestingly, a single gavage administration of 20 and 40 mg/kg RA significantly increased insulin-stimulated glucose transport activity by 223 and 286 pmol/mg/20 min, respectively, compared with SHAM rats. However, only a single gavage of 40 mg/kg RA increased the insulin-mediated glucose transport activity (the difference between basal and insulin-stimulated glucose transport activity) by 201 pmol/mg/20 min, P < 0.05 (Fig. 2). Moreover, this study found increased ERK1/2 activity in insulin-stimulated conditions compared with the ANG II-treated group, P < 0.05 (Fig. 3).