Effects of Losartan on Blood Pressure, Metabolic Alterations, and Vascular Reactivity in the Fructose-Induced Hypertensive Rat
Abstract Fructose feeding induces a moderate increase in blood pressure levels in normal rats that is associated with insulin resistance, hyperinsulinemia, and hypertriglyceridemia. The sympathetic nervous system seems to participate in the alterations of this model. To further explore the mechanisms underlying fructose-induced hypertension, the effects of the AT1 receptor antagonist losartan on blood pressure, insulin resistance, renal function, and vascular reactivity in mesenteric vascular beds were studied. Sprague-Dawley rats were fed for 4 weeks with diets containing 60% fructose or 60% starch (control), and half of each group received losartan (1 mg/kg per day) in the drinking water. Fructose-fed rats showed higher (P<.05) blood pressure levels and plasma concentrations of triglycerides and insulin than those of controls. Losartan treatment prevented both blood pressure elevation and hyperinsulinemia in fructose-fed rats but not elevation of plasma triglycerides. Plasma glucose and insulin levels in response to an oral glucose load were higher (P<.05) in fructose-fed rats than in controls. These exaggerated responses were prevented by losartan treatment. No differences in the constrictor responses of mesenteric vascular beds to KCl (60 μmol), angiotensin II (1 nmol), phenylephrine (10−5 mol/L), or endothelin-1 (10 pmol) were found between the two groups. Relaxing responses to acetylcholine or sodium nitroprusside in phenylephrine-precontracted mesenteric vascular beds and constrictor response to the nitric oxide synthesis inhibitor NG-nitro-l-arginine methyl ester (100 nmol) were comparable in both groups. Losartan blunted angiotensin II constriction and reduced (P<.05) responses to phenylephrine in all groups. In conclusion, these results suggest that angiotensin II plays an important role in the blood pressure elevation and in the insulin resistance induced by fructose feeding in rats. The mechanisms underlying these effects appear to be dependent on neither a vascular hyperreactivity to constrictor factors nor an endothelial dysfunction related to a decreased production of nitric oxide.
Several studies have shown an association between essential hypertension, insulin resistance, and alterations in the circulating lipidic profile, which has been conventionally termed syndrome X.1 2 Insulin resistance, hyperinsulinemia, and mild hypertension have been induced in normotensive rats by chronic high-sucrose or high-fructose feeding.3 4 The precise mechanisms by which hypertension develops in fructose-fed rats have not yet been clearly defined. Since somatostatin administration inhibited the hypertension induced by fructose feeding,5 it has been proposed that the rise in BP observed in this model is secondary to the development of hyperinsulinemia. It has been demonstrated that vanadyl sulfate, a drug able to decrease insulin levels in nondiabetic rats, prevented fructose-induced hyperinsulinemia and hypertension in rats.6 In addition, it has been suggested that an increase in sympathetic activity could account for the hypertension induced by fructose feeding.3 Furthermore, circulating catecholamines are elevated in sucrose-fed rats on a high salt diet, and it has been proposed that they contribute to hypertension through their vasoconstrictor and/or antinatriuretic properties.7
Angiotensin-converting enzyme inhibitors have been extensively shown to improve insulin sensitivity in hypertensive patients,8 9 thus suggesting a possible role of angiotensin II in the mechanisms of insulin resistance associated with hypertension. The role of the renin-angiotensin system in the pathogenesis of fructose-induced hypertension is unclear. Previous studies have shown nonelevated plasma renin activity or angiotensin II levels in this experimental model.10 In addition, endothelium-dependent and -independent relaxations as well as enhanced responsiveness to vasoconstrictors have been reported to be impaired in diabetic rats and in several models of hypertensive rats.11 12 These alterations have been proposed to be partially responsible for BP elevation and vascular remodeling.12 However, to our knowledge, vascular reactivity in resistance vessels from fructose-fed rats has not been studied to date.
To further explore the mechanisms underlying fructose-induced hypertension and insulin resistance in rats, the effects of the AT1 receptor antagonist losartan on BP, glycidic and lipidic circulating metabolic profiles, and insulin sensitivity were studied. In addition, renal function and vascular reactivity in MVB were studied, and the effects of losartan treatment were evaluated.
Thirty-two male Sprague-Dawley rats (weight, 120±3 g) were fed a diet (used as control) containing 60% vegetable starch, 11% fat, and 29% protein. After 2 weeks on this diet, half of the animals were randomly assigned to either the same control diet or a diet containing 60% fructose, 11% fat, and 29% protein for 4 additional weeks. Half of the animals of each group received the AT1 receptor antagonist losartan (1 mg/kg per day) in drinking water for this 4-week period of study. The electrolyte content of the control diet was 3.6 g/kg sodium and 10.8 g/kg potassium. The fructose diet contained 4.9 g/kg sodium and 10.8 g/kg potassium. The compositions of the diets were similar to that previously reported.3 Animals had free access to food and water and were maintained in a 12-hour light/dark cycle. All experiments were performed according to the guidelines for the ethical treatment of animals of the European Union. At the beginning and at the end of the 4-week period, systolic BP was estimated by a tail-cuff method, blood samples were obtained, and rats were housed in cages to collect 24-hour urine samples and to control food and water intakes.
Oral Glucose Tolerance Test
At the end of the 4-week period, plasma insulin and glucose concentrations were measured in response to an oral glucose tolerance test. Animals were fasted overnight and given only water to drink. On the morning of the test, a control blood sample (0.4 mL) was drawn from the tail, and each animal received an oral glucose load of 1 mL per 100 g body wt of a 50% (wt/vol) solution of glucose by oral gavage. Additional tail blood samples were drawn at 15 (0.4 mL), 30 (0.4 mL), 60 (0.1 mL), and 90 (0.4 mL) minutes after an oral glucose load. All samples, collected in micro–hematocrit tubes, were immediately centrifuged and stored at −20°C until the assay for plasma concentrations of glucose and insulin.
Contractile Responses in MVB
After rats were anesthetized and killed by decapitation, the superior mesenteric artery was isolated at its junction with the abdominal aorta and freed of fat and connective tissue. The mesentery was gently separated from the intestinal wall and perfused at constant flow (2 mL/min) with an oxygenated Krebs’ bicarbonate buffer at 37°C. Then the mesentery was removed from the animal and connected to a pressure transducer (model P23XL, Spectromed). Perfusion pressure was continuously recorded on a polygraph (7E, Grass Instrument) for the rest of the experiment. Constrictor or relaxant responses were measured as increases or decreases, respectively, of the perfusion pressure at a constant flow (2 mL/min). After an equilibration period of 90 minutes, the constrictor responses to boluses of KCl (60 μmol), angiotensin II (1 nmol), and endothelin-1 (10 pmol) were evaluated. After that, MVB were continuously perfused with phenylephrine (10−5 mol/L). When a steady submaximal contraction was reached, a dose-relaxation curve to acetylcholine (10−12 to 10−8 mol) was performed. After the last vasodilator response to acetylcholine, a single dose of the nitric oxide synthesis inhibitor L-NAME (100 nmol) was added. When a steady contraction to L-NAME was reached, vasodilator response to a single dose of sodium nitroprusside (10 nmol) was evaluated.
BP Measurement Procedure
Systolic BP was measured by the tail-cuff method with an electrosphygmograph (Narco Bio-Systems). At 10 days before the initiation of fructose feeding, rats were trained daily for the measurement of BP by the tail-cuff method. Each day, rats were placed (9 am) in their maintenance cages in a room at 28°C for 2 hours. Afterward, systolic BP was measured in unrestrained animals. Once the rats were considered to be trained and not susceptible to stress from the tail-cuff procedure, systolic BP measurements were performed. Basally and at week 4, systolic BP was measured on 2 consecutive days at the same time (11 am). Eight systolic BP measurements were carried out on each of these days in each animal, with the maximum and the minimum values being rejected. To validate the tail-cuff method for BP measurements, a catheter was implanted in the femoral artery of six animals treated with fructose for 4 weeks. Two days later, catheters were connected to a pressure transducer (model P23XL, Spectromed), and systolic BP was recorded on a polygraph (7E, Grass Instrument) for 60 minutes. The mean value of direct systolic BP (125±4 mm Hg) compared with the mean value of indirect measurements (130±5 mm Hg) showed a correlation of 96%.
Concentrations of sodium were measured in plasma and in urine samples by flame photometry (NAK-I, Pacisa). Colorimetric reactions assessed by commercial kits were used to determine plasma and urine levels of creatinine and plasma triglycerides (Medical Analysis Systems, Inc). Plasma insulin levels were measured by a radioimmunoassay method, and plasma glucose levels were determined by the colorimetric glucose oxidase reaction (DRG-Instruments GmbH).
All drugs (Sigma Chemical Co) and chemicals were dissolved in pyrogen-free deionized water to prepare buffer and stock solutions. Further dilutions were made into Krebs’ bicarbonate solution of the following composition (mmol/L): NaCl 118.5, KCl 4.7, CaCl2 2.8, KH2PO4 1.2, MgSO4 1.1, NaHCO3 25.0, and glucose 11.1.
Values are expressed as mean±SEM. Data were analyzed by ANOVA followed by a Newman-Keuls test; P<.05 was considered significant.
BP, Renal Function, and Metabolic Parameters
The Table⇓ shows values of BP, body weight, urine volume, sodium excretion, creatinine clearance, and plasma concentrations of triglycerides, glucose, and insulin in fructose-fed and control rats. After 4 weeks, fructose-fed rats showed increased systolic BP levels (P<.05) compared with controls. Losartan treatment prevented this BP elevation and had no effect on BP levels in control rats. Urine volume, sodium excretion, and creatinine clearance levels were comparable in fructose-fed and control rats and were not significantly affected by losartan treatment. Fructose-fed animals showed increased (P<.05) plasma levels of insulin and triglycerides compared with controls. Both groups of rats presented with similar fasting plasma glucose concentrations and gained the same amount of weight over the 4-week period of study. Losartan treatment did not affect plasma levels of glucose or triglycerides in either group but reduced elevated plasma insulin levels in fructose-fed rats.
Glucose Tolerance Test
Fig 1⇓ depicts plasma levels of glucose and insulin, basally and in response to an oral glucose load. Basal plasma insulin levels, but not glucose levels, were higher (P<.05) in fructose-fed rats than in controls (Fig 1⇓). Fifteen minutes after an oral glucose load, peak insulin and glucose plasma concentrations were found in all groups. Plasma glucose and insulin levels were higher (P<.05) in fructose-fed rats than in controls at 15, 30, and 60 minutes after an oral glucose load. In losartan-treated control and fructose-fed rats, comparable insulin and glucose plasma concentrations were observed basally and at 15, 30, and 90 minutes after an oral glucose load.
Vascular Reactivity in MVB
Constrictor responses to KCl, angiotensin II, phenylephrine, and endothelin-1 were similar in fructose-fed and control rats (Fig 2⇓). Treatment with losartan totally abolished responses to angiotensin II and reduced (P<.05) responses to phenylephrine in both groups (Fig 2⇓). Relaxing responses to acetylcholine (Fig 3⇓) and sodium nitroprusside (Fig 4⇓), as well as the constrictor response to L-NAME (Fig 5⇓), were comparable in control and fructose-fed rats and were not significantly affected by losartan treatment.
The present study shows that treatment with the AT1 receptor antagonist losartan prevents elevation in BP, hyperinsulinemia, and the exaggerated response to an oral glucose load induced by fructose feeding in rats. However, losartan did not affect elevated plasma triglycerides. This suggests the participation of angiotensin II in BP elevation and alterations in the glycidic metabolism observed in the fructose-fed rat.
Our results resemble those previously reported in rats and dogs,3 13 in which fructose feeding caused comparable degrees of hyperinsulinemia, hypertriglyceridemia, and hypertension without producing body weight gain or hyperglycemia. Concurrently, insulin resistance was developed,3 as is also shown by the impaired response to the glucose tolerance test observed in our study. This alteration has been reported to be a consequence of an impaired ability of insulin to suppress hepatic glucose output and stimulate glucose uptake in skeletal muscle and adipose tissue.14
The origin and mechanisms underlying fructose-induced hypertension and insulin resistance are not completely established. Several studies suggest that insulin resistance is not a mere consequence of hypertension, and others propose that hypertension is not caused by insulin resistance or hyperinsulinism.5 15 16 In humans, reports have indicated that both of these alterations could be secondary to other factors, such as increased skeletal muscle vascular resistance.17 Moreover, it has been shown that sucrose feeding increases norepinephrine excretion, turnover, and plasma concentrations and enhances sympathetic nerve responses in rats.5 7 18 19 Thus, a sympathetic overactivity has been involved in the pathogenesis of this model, since it may cause BP elevation and impairment of blood flow to skeletal muscle, which in turn would favor the development of insulin resistance.
Catecholamines appear not to be the only vasoconstrictor factors involved in the development of hypertension and insulin resistance in the fructose-fed rat. In fact, the present results suggest that angiotensin II could also be involved, because losartan treatment prevented BP elevation and improved insulin sensitivity. It should be noted that no significant change in plasma renin activity or circulating angiotensin levels has been observed in the fructose-fed rat.10 However, as in other experimental models, such as spontaneously hypertensive rats, tissue renin activity or angiotensin II production could be elevated without their systemic levels being altered.20 21 Thus, locally generated angiotensin II would induce a certain degree of peripheral vasoconstriction, which could contribute to the observed BP elevation and insulin resistance. Consequently, losartan might exert its beneficial effects by the suppression of vascular actions of angiotensin II via AT1 receptor antagonism. This would reduce elevated BP levels and restore a normal flow to the skeletal muscle, which in turn would facilitate glucose uptake.
In addition, it should be noted that losartan treatment not only abolished vasoconstrictor responses in MVB to angiotensin II but also reduced responses to phenylephrine in both fructose-fed and control rats. This indicates that at least part of the vasoconstrictor effect of catecholamines could be mediated by angiotensin II. Similar results have recently been shown in mesenteric arteries from spontaneously hypertensive and Wistar-Kyoto rats, in which losartan reduced the phenylephrine-induced decrease in diameter and flow.22 Therefore, since sympathetic overactivity seems to be present in the fructose-fed rat,3 a reduction of catecholamine constrictor response might be another mechanism by which losartan could contribute to prevent hypertension and improve insulin sensitivity.
Additional mechanisms such as vascular hyperreactivity to constrictor factors or endothelial dysfunction have been proposed to contribute to the development of hypertension and/or insulin resistance in diabetic rats and in other experimental models.11 12 Such alterations did not occur in our study since constrictor responses to KCl, angiotensin II, phenylephrine, or endothelin-1 were similar in fructose-fed and control animals. Moreover, a decreased nitric oxide production can be ruled out as a mechanism contributing to elevated BP because responses to acetylcholine and L-NAME were comparable in both groups. In contrast to that observed in diabetic rats,11 the present results indicate that the metabolic alterations induced by fructose feeding did not alter vasoactive responses in MVB. However, we cannot exclude the possibility that a longer period of hyperinsulinemia could affect vascular reactivity in fructose-fed rats.
The results also show that losartan prevented the elevation of insulin and BP in fructose-fed rats but did not influence elevated triglycerides. Hyperinsulinemia, insulin resistance, and hypertriglyceridemia have been proposed to have a common origin in sugar-fed rats.2 5 14 15 However, our results suggest that only BP elevation, hyperinsulinemia, and insulin resistance seem to be interrelated in the fructose-fed rat. To our knowledge, the possible mechanisms linking hypertriglyceridemia to insulin resistance and hypertension remain undefined. Sleder et al23 reported that both glucose and fructose feeding led to increased hepatic very-low-density lipoprotein–triglyceride synthesis and output but their removal from plasma was less efficient in fructose-fed rats. This difference was ascribed to the fact that fructose could not increase lipoprotein lipase activity, as does glucose. This characteristic of a fructose diet might account for the elevated plasma triglyceride levels after losartan treatment. In addition, in fructose-fed dogs hypertriglyceridemia preceded both hyperinsulinemia and hypertension.13 It appears that hypertriglyceridemia is more permanent than other metabolic alterations in fructose-fed rat and dogs. Consequently, it could be postulated that not all metabolic alterations observed in this experimental model are developed simultaneously or are due to the same mechanisms.
Finally, it should be mentioned that hyperinsulinism has also been associated with renal sodium retention,24 which could contribute to the development of hypertension. However, in the present study, in which renal excretory function was evaluated after 4 weeks of fructose feeding, we could not observe significant changes in urinary sodium excretion. In a previous study we observed increased BP values together with positive sodium balance during the first week of fructose feeding in rats, which returned to normal values by the second week.25 A moderate and transient sodium retention has also been described in fructose-fed dogs.13 Thus, in view of these results it could be postulated that sodium retention might be a triggering mechanism for the development of fructose-induced hypertension, but it appears that it is not a determining factor for the maintenance of this type of hypertension.
In conclusion, the present results suggest that angiotensin II plays an important role in both the BP elevation and insulin resistance induced by fructose feeding in rats. In contrast, the mechanisms underlying these effects appear to be dependent on neither a vascular hyperreactivity to constrictor factors nor an endothelial dysfunction related to a decreased production of nitric oxide.
Selected Abbreviations and Acronyms
|AT1||=||angiotensin II subtype 1|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|MVB||=||mesenteric vascular beds|
This study was supported by a grant from Comisión Interministerial de Ciencia y Tecnología (Spain) (SAF 95-1549-C02-01) and a medical school grant from Merck and Co, Inc, Whitehouse Station, NJ, USA. The authors thank Lucila Krauss and Antonio Carmona for their technical assistance, Anthony DeMarco for his editorial assistance, and Merck Pharmaceutical Co for kindly providing losartan.
- Received June 18, 1995.
- Revision received August 1, 1995.
- Accepted August 18, 1995.
Modan M, Halkin H, Almog S. Hyperinsulinemia: a link between hypertension, obesity and glucose intolerance. J Clin Invest. 1985;75:809-817.
Reaven GM. Role of insulin resistance in human disease. Diabetes. 1988;37:1595-1607.
Hwang I, Ho H, Hoffman BB, Reaven GM. Fructose-induced insulin resistance and hypertension in rats. Hypertension. 1987;10:512-516.
Buñag RD, Tomita T, Sasaki S. Chronic sucrose ingestion induced mild hypertension and tachycardia in rats. Hypertension. 1983;5:218-225.
Reaven GM, Ho H, Hoffmann BB. Somatostatin inhibition of fructose-induced hypertension. Hypertension. 1989;14:117-120.
Bhanot S, McNeill JH, Bryer-Ash M. Vanadyl sulfate prevents fructose-induced hyperinsulinemia and hypertension in rats. Hypertension. 1994;23:308-312.
Hsueh WA, Anderson PW. Hypertension, the endothelial cell, and the vascular complications of diabetes mellitus. Hypertension. 1992;20:253-263.
Lüscher TF, Bock HA, Yang Z, Diederich D. Endothelium-derived relaxing and contracting factors: perspectives in nephrology. Kidney Int. 1990;39:575-590.
Martínez FJ, Rizza RA, Romero JC. High-fructose feeding elicits insulin resistance, hyperinsulinism, and hypertension in normal mongrel dogs. Hypertension. 1994;23:456-463.
Facchini F, Chen YD, Clinkingbeard C, Jeppesen J, Reaven GM. Insulin resistance, hyperinsulinemia and dyslipidemia in nonobese individuals with a family history of hypertension. Am J Hypertens. 1992;5:694-699.
Baron AD, Brechtel-Hook G, Johnson A, Hardin D. Skeletal muscle blood flow: a possible link between insulin resistance and blood pressure. Hypertension. 1993;21:129-135.
Kost CK, Jackson EK. Enhanced renal angiotensin II subtype 1 receptor responses in the spontaneously hypertensive rat. Hypertension. 1993;21:420-431.
Qiu HY, Henrion D, Levy BI. Endogenous angiotensin II enhances phenylephrine-induced tone in hypertensive rats. Hypertension. 1994;24:317-321.
DeFronzo RA, Goldberg M, Agus Z. The effects of glucose and insulin on renal electrolyte transport. J Clin Invest. 1976;58:83-90.
Lahera V, Navarro J, García-Robles R, Rodicio JL, Romero JC, Ruilope LM. Antihypertensive effect of enalapril in the fructose-fed hypertensive rat. J Hypertens. 1992;10(suppl 4):S58. Abstract.