Chronic Insulin Infusion Normalizes Blood Pressure and the Gene Expressions of Angiotensin II Type 1 Receptor in Fructose-Fed Rats

127 Hypertens Res Vol.31 (2008) No.1 p.115-121 Original Article Chronic Insulin Infusion Normalizes Blood Pressure and the Gene Expressions of Angiotensin II Type 1 Receptor in Fructose-Fed Rats Tomoyasu FUKUI1), Tsutomu HIRANO1),Yuji SHIRAISHI1), Masaharu NAGASHIMA1), and Mitsuru ADACHI1) It remains open to debate whether hyperinsulinemia leads to the development of hypertension. We addressed this issue by investigating the effect of chronic insulin infusion on blood pressure and related parameters in hypertensive fructose-fed rats. Rats were given either normal chow or a fructose-rich diet, and insulin or saline was infused through mini-pumps in the same animals for 14 days. The chronic insulin infusion exerted no effect on the blood pressure of the chow-fed rats. Fructose feeding increased the blood pressure and levels of insulin, triglyceride and fatty acid. Insulin infusion augmented the hyperinsulinemia but normalized the blood pressure and plasma lipids. Plasma angiotensin II was elevated in the fructosefed rats, while insulin infusion left it unchanged. The expression of angiotensin II type 1 receptor (AT1R) mRNA was doubled in both the aorta and epididymal fat of the fructose-fed rats, while that of angiotensin II type 2 receptor (AT2R) was unaltered. Insulin infusion completely rectified the over-expression of the AT1R gene. Our findings indicate that chronic insulin infusion exacerbates hyperinsulinemia while normalizing blood pressure and the gene expressions of AT1R in insulin-resistant fructose-fed rats, suggesting that endogenous hyperinsulinemia caused by insulin resistance is associated with the development of hypertension, whereas exogenous hyperinsulinemia attenuates hypertension probably due to amelioration of insulin resistance. (Hypertens Res 2008; 31: 127–133) Key Words: insulin, fructose-fed rat, blood pressure, angiotensin II receptor Introduction Hypertension often develops in individuals with conditions associated with insulin resistance, such as obesity, type 2 diabetes, and metabolic syndrome. The hypertension accompanying insulin resistance is most often explained by hyperinsulinemia compensating for the insulin-resistant conditions. Insulin has been hypothesized to stimulate sympathetic nerve activity, sodium absorption in the renal tubules, and proliferation of smooth muscle cells in vessels (1–3). It remains unclear, however, whether insulin actually increases blood pressure (BP) in vivo. Indeed, patients with insulinoma are usually normotensive (4, 5). Hypertension, on the other hand, has possible links not with hyperinsulinemia, but with insulin resistance. Given that insulin resistance and hyperinsulinemia develop simultaneously, it can be difficult to ascertain the distinct role of hyperinsulinemia in the development of hypertension independently of insulin resistance. Rats fed a high-fructose diet serve well as an animal model for hypertension associated with insulin resistance and hyperinsulinemia (6–8). It remains to be determined, however, From the 1)First Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan. Address for Reprints: Tsutomu Hirano, M.D., First Department of Internal Medicine, Showa University School of Medicine, 1–5–8 Hatanodai, Shinagawa-ku, Tokyo 142–8666, Japan. E-mail: Received January 12, 2007; Accepted in revised form August 3, 2007. 128 Hypertens Res Vol. 31, No. 1 (2008) Table 1. General Profiles in Chow-Fed and Fructose-Fed Rats Infused with Saline or Insulin n SBP (mmHg) Pulse rate (/min) Food intake (g/day) Water intake (g/day) Initial body weight (g) Final body weight (g) Chow Chow with insulin Fructose Fructose with insulin 12 109±4 350±31 26.5±3.0 42.3±3.5 255±8 410±30 8 110±8 333±23 28.7±2.6 40.6±5.0 260±6 408±20 12 119±6* 369±14 18.5±3.7* 42.7±6.5 262±6 380±24* 12 108±12 361±11 25.8±5.5 43.5±4.5 258±10 406±28 SBP, systolic blood pressure. *p<0.05 chow-fed vs. fructose-fed. whether hyperinsulinemia is a causative factor behind the development of hypertension. Earlier reports have suggested that the renin-angiotensin system plays an important role in the development of hypertension associated with fructoseinduced hypertension accompanying insulin resistance/ hyperinsulinemia (9–11). Giacchetti et al. (12) reported elevations in the levels of angiotensin II (AII) type 1 receptor (AT1R) mRNA in fructose-fed rats, implicating AT1R mRNA as a possible contributor to fructose-induced hypertension. But it is unclear whether the elevated AT1R gene expression in fructose-fed rats is due to the development of hyperinsulinemia. Insulin has been reported to augment AT1R production in vitro (13), though it has yet to be determined whether hyperinsulinemia stimulates AT1R production in vivo. We investigated the effects of chronic insulin administration on BP and BP-related factors, such as the gene expressions of AII receptors, in hypertensive fructose-fed rats in order to determine whether further hyperinsulinemia influences hypertension and metabolic derangements induced by fructose feeding. Methods Rats Eight-week-old male Wistar rats (Charles River Japan, Tokyo, Japan) were divided into two groups and fed either standard rat chow containing 60% vegetable starch, 5% fat, and 24% protein (Oriental Yeast Co., Tokyo, Japan) or a fructose-rich chow containing 60% fructose, 5% fat, and 20% protein (Oriental Yeast Co.) for 21 days. Both diets contained 0.1% NaCl (w/w). Saline (0.9% NaCl) was used for both the control (saline anole) and insulin (solved with saline) infusions. During the last 14-day feeding period, the chow-fed and fructose-fed rats were each divided into subgroups and continuously infused with either human insulin (Novolin R, 6 U/24 h) solved with saline or saline vehicle alone at a rate of 4.5 μL/h by an osmotic mini-pump (Alzet Model 2ML2; Alza Corp., Palo Alto, USA) implanted subcutaneously. All rats were kept in individual cages on a rotating 12-h light–dark cycle with free access to food and water. The animals were fasted from 9:00 AM on the day of the experiment up to the commencement of the experiment at 2:00 PM. Drinking water remained available. All procedures were approved by the Institutional Animal Care and Use Committee of Showa University according to the Guidelines for the Care and Use of Laboratory Animals. BP and Heart Rate Measurement The systolic BP (SBP) and pulse rate were recorded in completely conscious rats using indirect tail-cuff equipment (BP Monitor MK-1030; Muromachi Kikai Co., Ltd., Tokyo, Japan) as described previously (14). The BP and pulse rate were each measured 5 times after pre-warming the rats on a 37°C plate for 20 min. The mean values were taken as the individual BP and pulse rate of each rat. Gene Expression of AT1R and AII Type 2 Receptor in the Aorta and Adipose Tissue Total RNA was extracted from the aortic tissue and epididymal fat pads using Isogen (Nippon Gene, Tokyo, Japan) according to the manufactu (...truncated)