Hyperinsulinemia superimposed on insulin resistance does not elevate blood pressure

Abstract

The role of hyperinsulinemia and insulin resistance in the development of hypertension is an area of much current interest. A central question that remains unanswered is whether exogenous hyperinsulinemia can elevate blood pressure (BP) in the presence of pre-existing insulin resistance. To examine this proposition, we studied the effects of chronic fructose feeding on plasma insulin levels and BP in insulin-resistant Zucker fatty rats and in lean (insulin-sensitive) controls. In addition, vascular responses to norepinephrine in aortae and mesenteric arteries were compared between groups. Zucker fatty rats were hyperinsulinemic, insulin-resistant, yet normotensive when compared with age-matched lean controls. Long term fructose feeding increased plasma insulin levels and BP in the lean group. Strikingly, the fatty rats remained refractory to fructose-induced increases in BP despite exaggeration of hyperinsulinemia. Vascular reactivity assessed in aortae and mesenteric arteries was comparable between groups. These data suggest that, in vivo, the mechanisms of hyperinsulinemia-induced hypertension are not operative in the face of pre-existing insulin resistance in obese Zucker rats

Hyperinsulinemia, hypertension, fructose feeding, Zucker fatty rats

For the past several years, there has been a growing interest in the hypothesis that resistance to the metabolic effects of insulin (insulin resistance) and compensatory hyperinsulinemia may contribute to increased blood pressure (BP) and essential hypertension, especially when associated with obesity.¹ A variety of clinical and experimental studies have yielded valuable pharmacological and mechanistic information in this regard.¹ In experimental models of hypertension, four key observations have served to strengthen this link and suggest possible causality.¹ First, treating insulin resistance in hypertension (with subsequent amelioration of hyperinsulinemia) lowers BP. Second, exogenous hyperinsulinemia (induced by fructose feeding) increases BP in rats; this rise is preceded by elevations in plasma insulin levels. Third, etiologically distinct models of hypertension exhibit similar changes in insulin sensitivity and BP. Finally, the vascular actions of insulin are altered in states of insulin resistance in favor of increased vascular tone and BP. These data suggest that insulin resistance and hyperinsulinemia may play important roles in the development and course of hypertension in rodent models of high BP. Because both hyperinsulinemia and insulin resistance have been proposed to independently modulate BP,¹ it is important to examine whether exogenous hyperinsulinemia superimposed on pre-existing insulin resistance elevates BP. This information will help to determine whether the hypertensinogenic effects of hyperinsulinemia are preserved (versus blunted) in states of whole body insulin resistance. To examine this proposition, we studied the effects of fructose-induced hyperinsulinemia on systolic blood pressure (SBP) in Zucker fatty (versus lean) rats. The Zucker fatty (fa/fa) rat is a well documented model of insulin resistance, obesity, and hyperinsulinemia.^2–4

Materials and Methods

Male Zucker lean (fa/−) and Zucker fatty (fa/fa) rats (7 weeks old) obtained from the Department of Physiology, University of British Columbia were divided into four experimental groups: lean untreated (n = 6), lean fructose-treated (n = 6), fatty untreated (n = 6), and fatty fructose-treated (n = 6). The experimental protocol was approved by the University of British Columbia Committee on Animal Care. After baseline measurements of insulin, glucose, and SBP, the rats in the treated groups were started on a fructose-enriched diet (66%) to induce hyperinsulinemia as described previously by Reaven et al and by others.^5–8 Systolic blood pressure was measured in conscious rats using the indirect tail-cuff method without external preheating.⁹ This method has been validated in our laboratory and closely approximates direct intra-arterial BP values (within 5 mm Hg). Biochemical measurements (5-h fasted glucose and insulin) were measured as described previously.^6–8 Following 5 weeks of treatment, plasma insulin levels, 5-h fasted glucose/insulin ratio (index of insulin sensitivity) and SBP were compared between groups using a two-way analysis of variance (ANOVA) followed by a Newman-Keuls test for post hoc comparisons. A P value of < .05 was set as the level of significance.

Vascular reactivity studies were performed in segments of aortae and superior mesenteric arteries from the four experimental groups. The tissues were suspended on wire hooks in isolated tissue baths containing modified Krebs-Ringer bicarbonate solution with the following composition (in mmol/L): NaCl (118), KCl (4.7), CaCl₂ (2.5), KH₂PO₄ (1.2), MgSO₄ (1.2), NaHCO₃ (25), dextrose (11.1), and calcium disodium edetate (0.026), maintained at 37°C, and oxygenated with 95% O₂ and 5% CO₂. Each ring was subjected to a resting tension determined to allow maximal force generation. After equilibration (60 min), isometric dose-response curves (DRC) to cumulative addition of norepinephrine (NE) were recorded. For each concentration, a plateau was obtained before the subsequent dose was added. Agonist pEC₅₀ values (−log EC₅₀) were calculated by nonlinear regression analysis of the DRC and used as an index of sensitivity.

Results

Fig. 1A and 1B depict the effects of fructose feeding on plasma insulin levels and BP in the four experimental groups. The Zucker fatty group was insulin resistant when compared with the lean controls (5-h fasted glucose/insulin ratio: 1.01 ± 0.17 v lean 7.52 ± 0.84, P < .05). Fructose feeding induced hyperinsulinemia and hypertension in the lean treated group (plasma insulin: 2.07 ± 0.3 v lean 1.15 ± 0.1 ng/mL, P < .05; SBP: 153 ± 4 v lean 131 ± 3 mm Hg, P < .05). Strikingly, in fatty rats, fructose-induced hyperinsulinemia failed to elevate BP (plasma insulin: 26.7 ± 5 v fatty untreated 9.3 ± 1 ng/mL, P < .05; SBP: 137 ± 3 v fatty untreated 133 ± 4 mm Hg, NS). Plasma glucose was similar among all four groups (Table 1).

Table 1

General characteristics at termination (16 weeks of age)

	Lean(n = 6)	Fatty(n = 6)	Lean-Fructose(n = 6)	Fatty-Fructose(n = 6)
Weight (g)	342 ± 1*	512 ± 21	324 ± 21*	554 ± 6†
Cholesterol (mmol/L)	2.4 ± 0.1*	6.1 ± 0.8	2.9 ± 0.2*	9.5 ± 0.3†
Triglycerides(mmol/L)	1.8 ± 0.1‡	8.9 ± 0.4	6.8 ± 0.6	7.1 ± 0.6
Glucose (5 h fasted,mmol/L)	7.4 ± 0.3	7.5 ± 0.2	7.7 ± 0.4	8.5 ± 0.3

^*

P < .05 different from fatty and fatty-fructose

^†

P < .05 different from lean, fatty, and lean-fructose

^‡

P < .05 different from fatty, lean-fructose, and fatty-fructose

Plasma insulin levels (ng/mL) (A, left and right panels) and SBP (mm Hg) (B) in the four experimental groups: Zucker lean-untreated (L, n = 6), Zucker lean fructose-treated (LF, n = 6), Zucker fatty-untreated (F, n = 6), and Zucker fatty fructose-treated (FF, n = 6). Data are expressed as mean ± SE. ^®P < .05, v L and F groups respectively; ^#P < .05, F v L, LF, and FF; *P = .05, v L, F, and FF.

Figure 1.

Figs. 2 and 3. depict the responses of aortae and mesenteric arteries to NE in the four experimental groups with intact endothelium. No difference in vascular reactivity to NE was observed between groups; the area under the curve (AUC) and the pEC₅₀ values were similar between groups in both aortae and mesenteric arteries, with endothelium intact (Table 2)

Dose-response curve of isolated superior mesenteric artery rings from the four experimental groups to norepinephrine with intact endothelium. Each point is represented as mean ± SE.

Figure 3.

Table 2

Vascular reactivity of aortae and mesenteric arteries to NE (intact endothelium) area under the curve (AUC) and pEC₅₀ values

	Aortae
	AUC	pEC50
Lean (n = 6)	226 ± 11	7.4 ± 0.1
Fatty (n = 6)	235 ± 14	7.6 ± 0.20
Lean-fructose (n = 5)	209 ± 6	7.3 ± 0.10
Fatty-fructose (n = 5)	213 ± 11	7.3 ± 0.10
	SuperiorMesenteric Artery
	AUC	pEC50
Lean (n = 6)	176 ± 12	6.8 ± 0.20
Fatty (n = 6)	201 ± 13	7.1 ± 0.20
Lean-fructose (n = 5)	166 ± 29	6.7 ± 0.40
Fatty-fructose (n = 5)	192 ± 23	7.1 ± 0.30

P > .05 among groups.

Dose-response curve of isolated thoracic aortae from the four experimental groups to norepinephrine with intact endothelium. Each point is represented as mean ± SE.

Figure 2.

Fructose feeding of Sprague-Dawley rats results in hyperinsulinemia and hypertension with a peak response at 3 weeks. The metabolic picture in the present study indicated no difference in the plasma insulin or glucose values after 3 v 7 weeks of fructose feeding (data not shown).

Discussion

The primary finding of this study is that exogenous hyperinsulinemia does not elevate BP when superimposed on insulin resistance. Furthermore, these data suggest that, in vivo, the mechanisms of hyperinsulinemia-induced hypertension are blunted in the face of whole body insulin resistance. These results have important implications for the insulin hypothesis of hypertension inasmuch as they show the resistance of Zucker fatty rats to fructose-induced hypertension.

A variety of mechanisms have been proposed to mediate fructose-induced hypertension in rats.¹ Through a variety of pharmacological studies, we have proposed that the pathogenesis of fructose-induced hypertension involves a close interrelationship between alterations in insulin action, vascular reactivity, and the sympathetic nervous system.¹ The sympathetic nervous system may be one of the primary systems activated in response to fructose feeding.⁶ Activation of this system may lead to the development of insulin resistance and compensatory hyperinsulinemia. Hyperinsulinemia may serve as a continual stimulus for sympathetic activation, thereby completing a vicious cycle of insulin resistance–hyperinsulinemia–sympathetic activation. Concurrently as well as independently, hyperinsulinemia may serve to increase endothelin-1 levels⁸ and to enhance the pressor response of resistance vasculature to sympathetic activation. In conjunction with increased endothelin-1 production, a loss of the vasodilatory actions of insulin (vascular insulin resistance) may serve to increase peripheral vascular resistance/tone and increase BP. Because perfusion per se is a determinant of insulin sensitivity,¹⁰ increased peripheral vascular resistance may reinforce the insulin-resistant state. Thus, if the cycle is broken by improving insulin sensitivity, blocking the effects of endothelin, antagonizing sympathetic activation, or promoting vasodilation, the net result is a decrease in BP.

Given this preamble, two questions arise from this study. First, why does fructose feeding (with the resultant hyperinsulinemia and insulin resistance) fail to increase BP in Zucker fatty rats? Although we cannot provide an unequivocal answer to this question, the most logical explanation would be that, in states of whole body insulin resistance, the mechanisms of hyperinsulinemia/insulin resistance–induced hypertension are blunted. Because hyperinsulinemia has been proposed to elevate BP through increasing endothelin-1 content (in mesenteric arteries), it is tempting to speculate that arteries from insulin-resistant fatty Zucker rats are refractory to this effect. Alternatively, a loss of the vasodepressor effects of insulin, as a consequence of insulin resistance, may tip the balance in favor of vasoconstriction.^11,12

The second important point that these data show is that, despite the presence of insulin resistance and hyperinsulinemia (at baseline), Zucker fatty rats remain normotensive when compared with their lean controls. Given the degree of insulin resistance and hyperinsulinemia noted in the untreated fatty Zucker group (plasma insulin levels 9.3 ± 1 v lean 1.15 ± 0.1 ng/mL, P < .0001), the absence of hypertension argues against a role of insulin resistance and hypertension in the development of high BP in this model. Whether insulin resistance and hyperinsulinemia are etiologically unimportant, or whether some unknown mechanism/factor affords protection from their hypertensinogenic effects, is an important question that remains unanswered at this time.

It is important to note that there is a discrepancy in the literature regarding the BP of Zucker fatty rats.² Only half of the published studies report modestly elevated BP, whereas the other half demonstrate a normotensive state consistent with the present study. The age of the fatty rat and the extent of renal damage appear to be key determinants of the BP response² and may help to explain the variability in the literature. By 6 months of age, the obese Zucker rat exhibits diminishing glomerular filtration rate and worsening renal function compared with those of lean animals.^3,13 The development of spontaneous glomeruloscerlosis is well documented in these rats, and it is possible that the expression of hypertension is dependent upon the manifestation of renal injury. In an important study by Kaskike et al,¹³ the effects of a high-salt diet on BP responses in fatty and lean Zucker rats were examined. The fatty rats remained normotensive and salt sensitive until the development of renal injury (at 8 months), suggesting that the kidney may play an important role in the development of high BP in this model. The primacy of renal failure in the etiology of hypertension in this model is further strengthened by studies,¹¹ including the present, that depict normal vascular reactivity in fatty versus lean rats.

In conclusion, fructose feeding does not elevate BP in Zucker fatty rats. This suggests that, in vivo, the mechanisms of hyperinsulinemia-induced hypertension are not operative in the face of pre-existing insulin resistance.