- Email: [email protected]
Effects of hyperinsulinemia on the regulation of regional blood flow and blood pressure in anesthetized dogs
AJH
1998;11:1232–1238
Effects of Hyperinsulinemia on the Regulation of Regional Blood Flow and Blood Pressure in Anesthetized Dogs Hemodynamic Role of Nitric Oxide Eduardo Villa, Rafael Garcı´a-Robles, and Juan Carlos Romero
The purpose of this study was to investigate whether acute hyperinsulinemia induces selective hemodynamic effects in the mesenteric, renal, and iliac vascular beds, and to determine whether nitric oxide (NO) plays a role in the regulation of blood flow and mean arterial pressure (MAP) during acute hyperinsulinism. In eight anesthetized dogs (Group A), the response to a hyperinsulinemic test was determined before and after NO inhibition, with L-nitro-arginine methyl esther (L-NAME), during the last 45 min of the experiment. In seven dogs (Group B), NO inhibition was induced before and maintained throughout hyperinsulinemia. In Group A, the hyperinsulinemic test did not alter MAP, but induced a significant reduction in both renal and mesenteric blood flow without a significant change in iliac blood flow. In contrast, the administration of L-NAME in Group B was
followed by a significant decrease in mesenteric, renal, and iliac blood flow, but mean arterial pressure remained unchanged. In this group, hyperinsulinemia instituted after the blockade of NO was followed by a significant elevation in blood pressure levels, concomitant with reductions in blood flow to the three vascular beds. In summary, acute hyperinsulinemia induced a redistribution of blood supply, which preserves skeletal muscle irrigation while reducing blood flow to the kidney. Nitric oxide participates in this redistribution because L-NAME infusion abolishes the compensatory influence on skeletal muscle blood flow. Am J Hypertens 1998;11:1232–1238 © 1998 American Journal of Hypertension, Ltd.
he strong interest in the characterization of the hemodynamic effects of insulin has been prompted primarily by the frequent association of insulin resistance with hypertension. Definite conclusions from early studies about the effect of insulin on blood pressure were precluded by paradoxical results where the acute sympathetic1 and
T
antinatriuretic2 effects of insulin contrasted with the failure of prolonged infusion of the hormone to produce hypertension.3–7 Furthermore, some patients with hyperinsulinemia do not exhibit a higher incidence of hypertension.8,9 The effects of insulin on blood pressure may be dependent on animal species. Elevation of circulating levels of insulin within the
Received October 31, 1997. Accepted April 27, 1998. From the Ramon y Cajal Institute, Madrid, Spain, and Department of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota. The present work was supported by grants from the Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, and Mayo Foundation, Rochester, Minnesota. Eduardo Villa
was a recipient of the Spanish DuPont-Pharma award and a postdoctoral fellow in the Department of Physiology, Mayo Clinic. Address correspondence and reprint requests to Dr J. C. Romero, Department of Physiology and Biophysics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Hypertension, L-nitro-arginine methyl ester, insulin resistance.
0895-7061/98/$19.00 PII S0895-7061(98)00133-2
AJH–OCTOBER 1998 –VOL. 11, NO. 10, PART 1
physiologic range induces sympathetic activation and hypertension in rats acutely10 and chronically.11 However, hyperinsulinemia did not increase blood pressure in dogs5–7 or humans,3,4 except when insulin was given at pharmacologic doses.1 These results lead to the general consensus that insulin produces very small effects on peripheral resistance.12–15 However, some observations in humans point to occasional insulininduced vasodilator effects.16,17 These effects were better defined in subsequent experiments that measured the effects of insulin on skeletal muscle vasculature.18 –21 The mechanism of function underlying such a vasodilator effect has never been clearly defined, although experimental observation has implicated b-adrenergic,22 Na1/K1pump,23,24 or Ca21-ATPase25,26 activation, along with increments in oxygen consumption. More recently the participation of endotheliumderived relaxing factor has been described in human studies, where the increase in forearm blood flow was abolished by the prior administration of NO synthesis inhibitor.27,28 This vasodilator effect of insulin has been explained as a physiologic mechanism to facilitate glucose and insulin delivery to the skeletal muscle. However, uncertainties still remain about the effect of this hormone on other vascular territories with different metabolic needs, accounting for large fractions of the cardiac output. Consequently, the purpose of our study was to test the hypothesis that acute hyperinsulinemia might evoke differential effects on mesenteric, renal, and iliac blood flow, and to determine whether nitric oxide could be a mediator of the vascular or systemic actions of insulin. Finally, we have explored the vascular and systemic actions of the neuropeptide somatostatin during normoinsulinemia. MATERIALS AND METHODS Surgical Procedure The study was performed in 20 mongrel dogs with an average body weight of 20.6 6 0.6 kg (range, 19.0 –21.5 kg). The animals were fed a standard pellet dog chow daily and had free access to water, except on the day before the experiment when all the dogs were fasted for 10 h. The animals were anesthetized with 30 mg/kg of sodium pentobarbital (intravenous) and ventilated according to the nomogram of Kleiman and Radford.29 The right femoral artery was catheterized for continuous blood pressure monitoring and blood sample collection, and the femoral vein was cannulated for continuous infusion of insulin and additional anesthetic. A catheter was inserted into the cephalic vein to infuse the solution for the insulin suppression test. Through a left flank incision, transonic flow probes (Transonic System Inc., Ithaca, NY) were placed in the mesenteric, renal, and iliac arterial segments proximal to the aorta for continuous blood flow monitoring in the three vascular beds. The left ureter was cannulated to collect urine
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samples. After the surgical procedure, bolus (1.6 mL/ kg) and continuous (0.03 mL/kg/min) infusions of 2.5% insulin were started, as was saline infusion at the same rate used during the test. The animals were allowed to stabilize for 45 to 60 min before the 15-min basal period. Insulin Suppression Test (IST) Hyperinsulinemia was achieved by infusing the following solutions for 180 min: 9 mg/kg/min dextrose as 30% solution in distilled water, 1 mU/kg/min porcine insulin as 20 mU/mL solution in 0.01% bovine serum albumin (BSA) saline, and 0.05 mg/kg/min somatostatin (added to the dextrose solution). Under these conditions, steady-state plasma levels for insulin and glucose were reached after 120 min and maintained for the rest of the experiment. From 120 to 180 min, arterial blood samples for analytical determinations were drawn every 10 min and urine samples were collected during the six 10-min periods (P1–P6) within 120 to 180 min. Experimental Design The animals were divided into three groups and matched for body weight. After the basal period (PB), the animals in Group A (n 5 8) underwent an IST that was maintained after the third h for an additional 45 min (180 –225 min), in which 10 mg/kg/min nitro-l-arginine methyl ester (L-NAME) was infused. During the last 15 min of this L-NAME infusion (PLN), the effects of partial NO blockade during a hyperinsulinemic state were evaluated. In Group B (n 5 7) after the PB, a 10-mg/kg/min LNAME infusion was started and maintained for the rest of the experiment. After 30 min of L-NAME infusion, a PLN was used to assess the effects of NO inhibition. Thereafter, an IST was performed in identical fashion to the previous group. This protocol was used to study the effects of hyperinsulinemia after NO blockade. The effects of somatostatin (SS) were assessed in Group C (n 5 5). After PB, the dogs were infused for 45 min with the same dose of SS used in groups A and B and a low dose of insulin (0.1 mU/kg/min). The last 15 min of the SS–low-dose insulin infusion was the period used for evaluation (PSS). The low dose of insulin was calculated previously to maintain insulin levels comparable to those during the basal period (normoinsulinemia). Blood samples for insulin, glucose, and Na1 and K1 levels were drawn at the end of each period, whereas blood samples for inulin were drawn at the midpoint of each period. Urine samples were collected at the end of the period. Analytical Determinations Plasma glucose (PG) levels were measured by the glucose oxidase method with a glucose analyzer (YSI 23A, Yellow Spring Instrument Co., Yellow Springs, OH), whereas plasma and urinary Na1 and K1 levels were determined
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FIGURE 1. Mean arterial pressure (MAP) and arterial blood flows in Group A (upper left and bottom left panel, respectively), and in Group B (upper right and lower right panel, respectively). Changes in MAP during hyperinsulinemic state are presented as average of periods P1 to P6. Values for mesenteric (MBF), renal (RBF), and iliac (IBF) blood flows are shown as percent compared to basal period.
with a flame photometer (Instrumentation Laboratory IL943). Plasma insulin (PI) was quantitated by a commercial RIA Kit (Linco Research Inc.). Finally, glomerular filtration rate (GFR) was calculated by measurement of inulin clearance; plasma and urine inulin concentrations were determined by the anthrone method.30 Statistical Analysis Where appropriate, withingroup analysis was carried out with paired Student’s t test or variance analysis for repeated measures followed by Dunnet’s test. Comparisons between the three groups were performed by ANOVA. A P , .05 was considered statistically significant. Values are expressed as means 6 SEM. RESULTS Group A During the basal period, the blood levels of glucose averaged 110.2 6 4.3 mg/dL and insulin av-
eraged 12.4 6 4.2 mU/mL, both levels in the normal range. During the IST, steady-state levels of plasma glucose averaged 141.4 6 11.5 mg/dL and insulin averaged 76.3 6 34.5 mU/mL; these were reached at 120 min and maintained for the rest of the experiment. Physiologic hyperinsulinemia was maintained during the seven evaluating periods (P1–PLN) with very small variations (range, 75.7–78.5 mU/mL), which went along with steady glycemia (range, 135.5–147.3 mg/dL). Figure 1A shows that under these conditions, mean arterial pressure (MAP) exhibited a slight upward trend, from 108.3 6 5.2 mm Hg at PB to 114.2 6 5.8 mm Hg during the periods P1 to P6. The administration of L-NAME produced a further elevation in the absolute average numbers of mean blood pressure, to 118.9 6 7.1 mm Hg. However, none of these changes achieved statistical significance when com-
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TABLE 1. GROUP C: SYSTEMIC AND RENAL PARAMETERS DURING SOMATOSTATIN AND LOW-DOSE INSULIN INFUSION
PB PSS
PG (mg/dL)
PI (mU/mL)
MAP (mm Hg)
MBF (% v PB)
RBF (% v PB)
IBF (% v PB)
106.3 6 4.5 104.0 6 3.6
11.2 6 2.3 9.2 6 1.2
107.7 6 7.2 111.6 6 9.0
100 84.0 6 3.6*
100 96.4 6 5.1
100 97.3 6 13.3
UV (mL/min)
UNaV (mEq/min)
GFR (mL/min)
0.45 6 0.4 109.0 6 71.7 0.85 6 0.8 95.6 6 58.9
33.5 6 6.2 35.8 6 9.3
Values are means 6 SEM; n 5 5 experiments. PG, plasma glucose; PI, plasma insulin; MAP, mean arterial pressure; MBF, mesenteric blood flow; RBF, renal blood flow; IBF, iliac blood flow; PB, basal period; UV, urine flow rate; UNaV, sodium excretion; GFR, glomerular filtration rate; PSS, somatostatin period. * P , .05 v PB.
pared with the values recorded during the control period. Figure 1B shows the changes in blood flow induced by hyperinsulinemia in the three vascular beds. It can be seen that blood supply to the mesenteric and renal territories experienced a significant decrease, whereas blood flow to the iliac was maintained. The administration of L-NAME after the last period was followed by a significant fall in the iliac and renal blood flow, whereas no changes were observed in the mesenteric flow. No significant changes, from the following basal values, were observed in either GFR (30.0 6 9.3 mL/ min), urinary flow (0.12 6 0.11 mL/min), or sodium excretion (33.45 6 35.2 mEq/min) during the experimental periods. Group B Basal values of mean arterial pressure (108 6 5.8 mm Hg), GFR (36.7 6 10.3 mL/min), urinary flow (0.12 6 0.1 mL/min), and sodium excretion (40.5 6 35.5 mEq/min) matched the levels obtained for those parameters in Group A for the same period. In addition, plasma levels of glucose (109.3 6 5.9 mg/dL) and insulin (12.9 6 3.1 mU/mL) were also similar to the corresponding values of Group A. None of these parameters significantly changed during the L-NAME period, although slight upward trends were recorded, in mean arterial pressure from 108.0 6 5.8 to 118.8 6 8.7 mm Hg, and for GFR from 36.7 6 10.3 to 43.2 6 19.6 mL/min. Hyperinsulinemia achieved during the IST was steady during all the periods, with values comparable to those obtained in Group A during the P1 to P6 periods (average, 76.3 6 34.5 in Group A versus 79.2 6 25.7 mU/mL in Group B). Glycemic response (average, 148.9 6 13.4 mg/dL) was also comparable to that achieved in Group A. This indicates that the differences between the two groups were not related to unmatched levels of glycemia or insulinemia. As shown in Figure 1D, the administration of L-NAME induced a significant decrease in the three arterial flows, which were further accentuated during hyperinsulinemia. These levels of blood flow remained unchanged until the end of the experiment. It should be
noted that hyperinsulinemia induced a significant increase in mean arterial pressure, from 118.8 6 8.7 mm Hg, recorded during the PLN preceding period, to 132.5 6 6.5 mm Hg during hyperinsulinemia (P , .05). GFR was slightly increased during the administration of L-NAME, whereas a significant decrease, from 43.2 6 19.6 to 23.2 6 5.6, was observed during hyperinsulinemia (P , .05 v PB and PLN). This fall may be secondary to the relevant fall in RBF. Group C Basal values of the animals included in this group were comparable to those of Groups A and B. When somatostatin was infused during the maintenance of fasting insulin levels (basal, 11.2 6 2.3 mU/ mL; PSS, 9.2 6 1.2 mU/mL), a significant 16% decrease in mesenteric blood flow (MBF) (P , .05) was observed. The rest of the parameters remained statistically unaltered under these conditions (Table 1). DISCUSSION The major observation in this study is that hyperinsulinemia, induced by an IST, failed to evoke any significant change in blood flow to the skeletal muscle, whereas the blood flow to the superior mesenteric and renal arteries was significantly decreased (Figure 1B) in anesthetized dogs. The vasoconstriction of these two important vascular beds was not sufficient to increase mean arterial pressure. Moreover, under these conditions, it would appear that NO was at least partially responsible for both maintaining iliac blood flow and minimizing the renal vasoconstriction, because the administration of L-NAME at the end of period 6 was followed by a pronounced fall of blood flow in these vascular territories. This suggests that the primary action of insulin in the iliac and renal arteries consists of vasoconstriction, which is ameliorated by the concomitant formation of NO. This assumption is strongly supported by the findings in Group B, which received a continuous intravenous infusion of L-NAME before inducing hyperinsulinemia. In these animals, the initial inhibition of the NO synthesis produced a significant fall in the basal levels of blood flow in the three vascular territories, suggest-
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ing that continuous production of this endothelial factor helps to maintain a certain degree of vasodilation. Further induction of hyperinsulinemia after L-NAME was followed by a uniform 15% to 20% fall of blood flow in all vascular beds, which was parallel to a significant rise in MAP. Our results on the lack of effect of hyperinsulinemia on the iliac vascular bed differ from some studies performed in humans by other investigations, which showed that insulin produces a significant vasodilation in the skeletal muscle18 –21; however, there is an equal amount of evidence reporting that insulin fails to induce any significant vascular change.12–15,31 Regardless of the possibility that interspecific or methodologic differences among the studies can account for this discrepancy, it seems that insulin-mediated vasodilation might help facilitate insulin delivery to the skeletal muscle and assure an adequate glucose uptake. Although the mechanism implicated in this vasodilation remains ill-defined,32 recent studies point to an increased production or release of NO as the major candidate responsible for this action, because skeletal muscle vasodilation was prevented by the previous inhibition of NO.27,28 In agreement with this hypothesis, in our study L-NAME infusion during hyperinsulinemia induced vasoconstriction (Group A), and hyperinsulinism instituted after NO blockade in Group B produced an additional reduction in the three blood flows. On the other hand, little attention has been paid to the effects of insulin in other vascular beds, such as the kidney, where facilitation of glucose uptake is not pivotal. Some studies have suggested a minor role for this hormone in the regulation of renal vascular tone. Chronic intrarenal infusion of insulin for 7 days failed to produce remarkable changes in effective RBF in uninephrectomized dogs.33 In addition, no significant change in RBF was observed during acute euglycemic hyperinsulinemia in humans.34 In our model, physiologic hyperinsulinemia induced a marked reduction of the RBF in Group A. At this point, it should be noted that the vascular tone of the mesenteric territory was not affected only by L-NAME, but also by the administration of somatostatin. In fact, in Group C, the mesenteric blood flow decreased by 16%, whereas the renal and iliac blood flow changed only minimally. The reduction induced by somatostatin in the MBF was comparable to that seen during hyperinsulinemia (Group A), thus indicating that hyperinsulinism does not influence MBF. This finding, together with the slight elevation in IBF, and the reduction in RBF attributable to hyperinsulinemia, raises the possibility that insulin could exert selective vascular effects in different vascular beds. Specific alterations in regional blood flows by the infusion of high doses of insulin (4 and 8 mU/kg/min)
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has been reported in the literature.22 In this study, skeletal muscle flow increased, whereas adrenal flow remained unchanged, and renal and splanchnic flows were reduced. In addition, insulin has been reported to induce a differential regulation between systemic and skeletal muscle vascular resistance.35 The mechanisms responsible for these different responses have been poorly evaluated. Hyperinsulinemia might selectively regulate production or release of vasodilator factors in different vascular beds. Nitric oxide has been shown to participate in the regulation of the blood supply to the skeletal muscle and the kidney, but the basal rate of NO synthesis appears to be different in different vascular beds. In fact, in our study the systemic infusion of L-NAME induced a variable degree of blood flow reduction in the three arteries (29% RBF, 216% MBF, and 219% IBF during PLN in Group B). It is then conceivable that the stimulation of NO synthesis may not uniformly affect vascular resistances in all vascular beds. A second possibility is the existence of local differences in vascular reactivity to vasoconstrictor stimuli. Insulin has been reported to enhance the expression and circulating levels of endothelin-1.36 An elevation in plasma norepinephrine concentrations in response to acute hyperinsulinemia was described,1 and subsequently confirmed by other authors with even lower doses of insulin.3,21 In addition, insulin has been reported to cause increased cardiovascular reactivity to the pressor effects of norepinephrine,37 although infusion of norepinephrine during a hyperinsulinemic state failed to cause any alteration in human forearm blood flow.28 These controversial data may reinforce the existence of vascular heterogeneity in the number or affinity of receptors for vasoconstrictor factors. A final possibility consists of the combination of the two previous mechanisms. A specific balance between insulin-induced vasodilation and vasoconstriction would determine net hemodynamic outcome in each vascular bed. The last aim of our study was to investigate the effects of physiologic hyperinsulinemia on blood pressure. In our model, hyperinsulinemia per se did not induce any significant change in MAP, in agreement with the large body of previous evidence in humans and dogs.3–7,27,28,37 However, when partial NO inhibition preceded and paralleled the same level of hyperinsulinemia, a significant rise in MAP was observed. In this line, NO blockade has been recently reported in humans to impair insulin-mediated skeletal muscle vasodilation, associated with an increase in mean arterial pressure, during an euglycemic hyperinsulinemic clamp.28 These data suggest the possibility that physiologic hyperinsulinemia may be a pressor mechanism in situations of endothelial derangement related to a defective NO system. This hypothesis might rec-
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oncile the controversy between lack of effect of acute and short-term hyperinsulinemism in the genesis of hypertension,3–7,38 and the epidemiologic correlation between hypertension, hyperinsulinemia, and insulin resistance.39,40
16.
Creager MS, Liang CS, Coffman JD: Beta adrenergic mediated vasodilator response to insulin in the human forearm. J Pharmacol Exper Ther 1985;235:709 –714.
17.
Gelfand RA, Barret EJ: Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest 1987;80:1– 6.
ACKNOWLEDGMENTS
18.
Baron AD, Brechtel-Hook G, Johnson A, et al: Skeletal muscle blood flow. A possible link between insulin resistance and blood pressure. Hypertension 1993;21: 129 –135.
19.
Jamerson KA, Julius S, Gudbrandsson O, et al: Reflex sympathetic activation induces acute insulin resistance in the human forearm. Hypertension 1993;21:618 – 623.
In memoriam Mary Olsen. We are indebted to Rodney Bolterman for excellent technical assistance.
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1998;11:1232–1238
Effects of Hyperinsulinemia on the Regulation of Regional Blood Flow and Blood Pressure in Anesthetized Dogs Hemodynamic Role of Nitric Oxide Eduardo Villa, Rafael Garcı´a-Robles, and Juan Carlos Romero
The purpose of this study was to investigate whether acute hyperinsulinemia induces selective hemodynamic effects in the mesenteric, renal, and iliac vascular beds, and to determine whether nitric oxide (NO) plays a role in the regulation of blood flow and mean arterial pressure (MAP) during acute hyperinsulinism. In eight anesthetized dogs (Group A), the response to a hyperinsulinemic test was determined before and after NO inhibition, with L-nitro-arginine methyl esther (L-NAME), during the last 45 min of the experiment. In seven dogs (Group B), NO inhibition was induced before and maintained throughout hyperinsulinemia. In Group A, the hyperinsulinemic test did not alter MAP, but induced a significant reduction in both renal and mesenteric blood flow without a significant change in iliac blood flow. In contrast, the administration of L-NAME in Group B was
followed by a significant decrease in mesenteric, renal, and iliac blood flow, but mean arterial pressure remained unchanged. In this group, hyperinsulinemia instituted after the blockade of NO was followed by a significant elevation in blood pressure levels, concomitant with reductions in blood flow to the three vascular beds. In summary, acute hyperinsulinemia induced a redistribution of blood supply, which preserves skeletal muscle irrigation while reducing blood flow to the kidney. Nitric oxide participates in this redistribution because L-NAME infusion abolishes the compensatory influence on skeletal muscle blood flow. Am J Hypertens 1998;11:1232–1238 © 1998 American Journal of Hypertension, Ltd.
he strong interest in the characterization of the hemodynamic effects of insulin has been prompted primarily by the frequent association of insulin resistance with hypertension. Definite conclusions from early studies about the effect of insulin on blood pressure were precluded by paradoxical results where the acute sympathetic1 and
T
antinatriuretic2 effects of insulin contrasted with the failure of prolonged infusion of the hormone to produce hypertension.3–7 Furthermore, some patients with hyperinsulinemia do not exhibit a higher incidence of hypertension.8,9 The effects of insulin on blood pressure may be dependent on animal species. Elevation of circulating levels of insulin within the
Received October 31, 1997. Accepted April 27, 1998. From the Ramon y Cajal Institute, Madrid, Spain, and Department of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota. The present work was supported by grants from the Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, and Mayo Foundation, Rochester, Minnesota. Eduardo Villa
was a recipient of the Spanish DuPont-Pharma award and a postdoctoral fellow in the Department of Physiology, Mayo Clinic. Address correspondence and reprint requests to Dr J. C. Romero, Department of Physiology and Biophysics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Hypertension, L-nitro-arginine methyl ester, insulin resistance.
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physiologic range induces sympathetic activation and hypertension in rats acutely10 and chronically.11 However, hyperinsulinemia did not increase blood pressure in dogs5–7 or humans,3,4 except when insulin was given at pharmacologic doses.1 These results lead to the general consensus that insulin produces very small effects on peripheral resistance.12–15 However, some observations in humans point to occasional insulininduced vasodilator effects.16,17 These effects were better defined in subsequent experiments that measured the effects of insulin on skeletal muscle vasculature.18 –21 The mechanism of function underlying such a vasodilator effect has never been clearly defined, although experimental observation has implicated b-adrenergic,22 Na1/K1pump,23,24 or Ca21-ATPase25,26 activation, along with increments in oxygen consumption. More recently the participation of endotheliumderived relaxing factor has been described in human studies, where the increase in forearm blood flow was abolished by the prior administration of NO synthesis inhibitor.27,28 This vasodilator effect of insulin has been explained as a physiologic mechanism to facilitate glucose and insulin delivery to the skeletal muscle. However, uncertainties still remain about the effect of this hormone on other vascular territories with different metabolic needs, accounting for large fractions of the cardiac output. Consequently, the purpose of our study was to test the hypothesis that acute hyperinsulinemia might evoke differential effects on mesenteric, renal, and iliac blood flow, and to determine whether nitric oxide could be a mediator of the vascular or systemic actions of insulin. Finally, we have explored the vascular and systemic actions of the neuropeptide somatostatin during normoinsulinemia. MATERIALS AND METHODS Surgical Procedure The study was performed in 20 mongrel dogs with an average body weight of 20.6 6 0.6 kg (range, 19.0 –21.5 kg). The animals were fed a standard pellet dog chow daily and had free access to water, except on the day before the experiment when all the dogs were fasted for 10 h. The animals were anesthetized with 30 mg/kg of sodium pentobarbital (intravenous) and ventilated according to the nomogram of Kleiman and Radford.29 The right femoral artery was catheterized for continuous blood pressure monitoring and blood sample collection, and the femoral vein was cannulated for continuous infusion of insulin and additional anesthetic. A catheter was inserted into the cephalic vein to infuse the solution for the insulin suppression test. Through a left flank incision, transonic flow probes (Transonic System Inc., Ithaca, NY) were placed in the mesenteric, renal, and iliac arterial segments proximal to the aorta for continuous blood flow monitoring in the three vascular beds. The left ureter was cannulated to collect urine
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samples. After the surgical procedure, bolus (1.6 mL/ kg) and continuous (0.03 mL/kg/min) infusions of 2.5% insulin were started, as was saline infusion at the same rate used during the test. The animals were allowed to stabilize for 45 to 60 min before the 15-min basal period. Insulin Suppression Test (IST) Hyperinsulinemia was achieved by infusing the following solutions for 180 min: 9 mg/kg/min dextrose as 30% solution in distilled water, 1 mU/kg/min porcine insulin as 20 mU/mL solution in 0.01% bovine serum albumin (BSA) saline, and 0.05 mg/kg/min somatostatin (added to the dextrose solution). Under these conditions, steady-state plasma levels for insulin and glucose were reached after 120 min and maintained for the rest of the experiment. From 120 to 180 min, arterial blood samples for analytical determinations were drawn every 10 min and urine samples were collected during the six 10-min periods (P1–P6) within 120 to 180 min. Experimental Design The animals were divided into three groups and matched for body weight. After the basal period (PB), the animals in Group A (n 5 8) underwent an IST that was maintained after the third h for an additional 45 min (180 –225 min), in which 10 mg/kg/min nitro-l-arginine methyl ester (L-NAME) was infused. During the last 15 min of this L-NAME infusion (PLN), the effects of partial NO blockade during a hyperinsulinemic state were evaluated. In Group B (n 5 7) after the PB, a 10-mg/kg/min LNAME infusion was started and maintained for the rest of the experiment. After 30 min of L-NAME infusion, a PLN was used to assess the effects of NO inhibition. Thereafter, an IST was performed in identical fashion to the previous group. This protocol was used to study the effects of hyperinsulinemia after NO blockade. The effects of somatostatin (SS) were assessed in Group C (n 5 5). After PB, the dogs were infused for 45 min with the same dose of SS used in groups A and B and a low dose of insulin (0.1 mU/kg/min). The last 15 min of the SS–low-dose insulin infusion was the period used for evaluation (PSS). The low dose of insulin was calculated previously to maintain insulin levels comparable to those during the basal period (normoinsulinemia). Blood samples for insulin, glucose, and Na1 and K1 levels were drawn at the end of each period, whereas blood samples for inulin were drawn at the midpoint of each period. Urine samples were collected at the end of the period. Analytical Determinations Plasma glucose (PG) levels were measured by the glucose oxidase method with a glucose analyzer (YSI 23A, Yellow Spring Instrument Co., Yellow Springs, OH), whereas plasma and urinary Na1 and K1 levels were determined
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FIGURE 1. Mean arterial pressure (MAP) and arterial blood flows in Group A (upper left and bottom left panel, respectively), and in Group B (upper right and lower right panel, respectively). Changes in MAP during hyperinsulinemic state are presented as average of periods P1 to P6. Values for mesenteric (MBF), renal (RBF), and iliac (IBF) blood flows are shown as percent compared to basal period.
with a flame photometer (Instrumentation Laboratory IL943). Plasma insulin (PI) was quantitated by a commercial RIA Kit (Linco Research Inc.). Finally, glomerular filtration rate (GFR) was calculated by measurement of inulin clearance; plasma and urine inulin concentrations were determined by the anthrone method.30 Statistical Analysis Where appropriate, withingroup analysis was carried out with paired Student’s t test or variance analysis for repeated measures followed by Dunnet’s test. Comparisons between the three groups were performed by ANOVA. A P , .05 was considered statistically significant. Values are expressed as means 6 SEM. RESULTS Group A During the basal period, the blood levels of glucose averaged 110.2 6 4.3 mg/dL and insulin av-
eraged 12.4 6 4.2 mU/mL, both levels in the normal range. During the IST, steady-state levels of plasma glucose averaged 141.4 6 11.5 mg/dL and insulin averaged 76.3 6 34.5 mU/mL; these were reached at 120 min and maintained for the rest of the experiment. Physiologic hyperinsulinemia was maintained during the seven evaluating periods (P1–PLN) with very small variations (range, 75.7–78.5 mU/mL), which went along with steady glycemia (range, 135.5–147.3 mg/dL). Figure 1A shows that under these conditions, mean arterial pressure (MAP) exhibited a slight upward trend, from 108.3 6 5.2 mm Hg at PB to 114.2 6 5.8 mm Hg during the periods P1 to P6. The administration of L-NAME produced a further elevation in the absolute average numbers of mean blood pressure, to 118.9 6 7.1 mm Hg. However, none of these changes achieved statistical significance when com-
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TABLE 1. GROUP C: SYSTEMIC AND RENAL PARAMETERS DURING SOMATOSTATIN AND LOW-DOSE INSULIN INFUSION
PB PSS
PG (mg/dL)
PI (mU/mL)
MAP (mm Hg)
MBF (% v PB)
RBF (% v PB)
IBF (% v PB)
106.3 6 4.5 104.0 6 3.6
11.2 6 2.3 9.2 6 1.2
107.7 6 7.2 111.6 6 9.0
100 84.0 6 3.6*
100 96.4 6 5.1
100 97.3 6 13.3
UV (mL/min)
UNaV (mEq/min)
GFR (mL/min)
0.45 6 0.4 109.0 6 71.7 0.85 6 0.8 95.6 6 58.9
33.5 6 6.2 35.8 6 9.3
Values are means 6 SEM; n 5 5 experiments. PG, plasma glucose; PI, plasma insulin; MAP, mean arterial pressure; MBF, mesenteric blood flow; RBF, renal blood flow; IBF, iliac blood flow; PB, basal period; UV, urine flow rate; UNaV, sodium excretion; GFR, glomerular filtration rate; PSS, somatostatin period. * P , .05 v PB.
pared with the values recorded during the control period. Figure 1B shows the changes in blood flow induced by hyperinsulinemia in the three vascular beds. It can be seen that blood supply to the mesenteric and renal territories experienced a significant decrease, whereas blood flow to the iliac was maintained. The administration of L-NAME after the last period was followed by a significant fall in the iliac and renal blood flow, whereas no changes were observed in the mesenteric flow. No significant changes, from the following basal values, were observed in either GFR (30.0 6 9.3 mL/ min), urinary flow (0.12 6 0.11 mL/min), or sodium excretion (33.45 6 35.2 mEq/min) during the experimental periods. Group B Basal values of mean arterial pressure (108 6 5.8 mm Hg), GFR (36.7 6 10.3 mL/min), urinary flow (0.12 6 0.1 mL/min), and sodium excretion (40.5 6 35.5 mEq/min) matched the levels obtained for those parameters in Group A for the same period. In addition, plasma levels of glucose (109.3 6 5.9 mg/dL) and insulin (12.9 6 3.1 mU/mL) were also similar to the corresponding values of Group A. None of these parameters significantly changed during the L-NAME period, although slight upward trends were recorded, in mean arterial pressure from 108.0 6 5.8 to 118.8 6 8.7 mm Hg, and for GFR from 36.7 6 10.3 to 43.2 6 19.6 mL/min. Hyperinsulinemia achieved during the IST was steady during all the periods, with values comparable to those obtained in Group A during the P1 to P6 periods (average, 76.3 6 34.5 in Group A versus 79.2 6 25.7 mU/mL in Group B). Glycemic response (average, 148.9 6 13.4 mg/dL) was also comparable to that achieved in Group A. This indicates that the differences between the two groups were not related to unmatched levels of glycemia or insulinemia. As shown in Figure 1D, the administration of L-NAME induced a significant decrease in the three arterial flows, which were further accentuated during hyperinsulinemia. These levels of blood flow remained unchanged until the end of the experiment. It should be
noted that hyperinsulinemia induced a significant increase in mean arterial pressure, from 118.8 6 8.7 mm Hg, recorded during the PLN preceding period, to 132.5 6 6.5 mm Hg during hyperinsulinemia (P , .05). GFR was slightly increased during the administration of L-NAME, whereas a significant decrease, from 43.2 6 19.6 to 23.2 6 5.6, was observed during hyperinsulinemia (P , .05 v PB and PLN). This fall may be secondary to the relevant fall in RBF. Group C Basal values of the animals included in this group were comparable to those of Groups A and B. When somatostatin was infused during the maintenance of fasting insulin levels (basal, 11.2 6 2.3 mU/ mL; PSS, 9.2 6 1.2 mU/mL), a significant 16% decrease in mesenteric blood flow (MBF) (P , .05) was observed. The rest of the parameters remained statistically unaltered under these conditions (Table 1). DISCUSSION The major observation in this study is that hyperinsulinemia, induced by an IST, failed to evoke any significant change in blood flow to the skeletal muscle, whereas the blood flow to the superior mesenteric and renal arteries was significantly decreased (Figure 1B) in anesthetized dogs. The vasoconstriction of these two important vascular beds was not sufficient to increase mean arterial pressure. Moreover, under these conditions, it would appear that NO was at least partially responsible for both maintaining iliac blood flow and minimizing the renal vasoconstriction, because the administration of L-NAME at the end of period 6 was followed by a pronounced fall of blood flow in these vascular territories. This suggests that the primary action of insulin in the iliac and renal arteries consists of vasoconstriction, which is ameliorated by the concomitant formation of NO. This assumption is strongly supported by the findings in Group B, which received a continuous intravenous infusion of L-NAME before inducing hyperinsulinemia. In these animals, the initial inhibition of the NO synthesis produced a significant fall in the basal levels of blood flow in the three vascular territories, suggest-
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ing that continuous production of this endothelial factor helps to maintain a certain degree of vasodilation. Further induction of hyperinsulinemia after L-NAME was followed by a uniform 15% to 20% fall of blood flow in all vascular beds, which was parallel to a significant rise in MAP. Our results on the lack of effect of hyperinsulinemia on the iliac vascular bed differ from some studies performed in humans by other investigations, which showed that insulin produces a significant vasodilation in the skeletal muscle18 –21; however, there is an equal amount of evidence reporting that insulin fails to induce any significant vascular change.12–15,31 Regardless of the possibility that interspecific or methodologic differences among the studies can account for this discrepancy, it seems that insulin-mediated vasodilation might help facilitate insulin delivery to the skeletal muscle and assure an adequate glucose uptake. Although the mechanism implicated in this vasodilation remains ill-defined,32 recent studies point to an increased production or release of NO as the major candidate responsible for this action, because skeletal muscle vasodilation was prevented by the previous inhibition of NO.27,28 In agreement with this hypothesis, in our study L-NAME infusion during hyperinsulinemia induced vasoconstriction (Group A), and hyperinsulinism instituted after NO blockade in Group B produced an additional reduction in the three blood flows. On the other hand, little attention has been paid to the effects of insulin in other vascular beds, such as the kidney, where facilitation of glucose uptake is not pivotal. Some studies have suggested a minor role for this hormone in the regulation of renal vascular tone. Chronic intrarenal infusion of insulin for 7 days failed to produce remarkable changes in effective RBF in uninephrectomized dogs.33 In addition, no significant change in RBF was observed during acute euglycemic hyperinsulinemia in humans.34 In our model, physiologic hyperinsulinemia induced a marked reduction of the RBF in Group A. At this point, it should be noted that the vascular tone of the mesenteric territory was not affected only by L-NAME, but also by the administration of somatostatin. In fact, in Group C, the mesenteric blood flow decreased by 16%, whereas the renal and iliac blood flow changed only minimally. The reduction induced by somatostatin in the MBF was comparable to that seen during hyperinsulinemia (Group A), thus indicating that hyperinsulinism does not influence MBF. This finding, together with the slight elevation in IBF, and the reduction in RBF attributable to hyperinsulinemia, raises the possibility that insulin could exert selective vascular effects in different vascular beds. Specific alterations in regional blood flows by the infusion of high doses of insulin (4 and 8 mU/kg/min)
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has been reported in the literature.22 In this study, skeletal muscle flow increased, whereas adrenal flow remained unchanged, and renal and splanchnic flows were reduced. In addition, insulin has been reported to induce a differential regulation between systemic and skeletal muscle vascular resistance.35 The mechanisms responsible for these different responses have been poorly evaluated. Hyperinsulinemia might selectively regulate production or release of vasodilator factors in different vascular beds. Nitric oxide has been shown to participate in the regulation of the blood supply to the skeletal muscle and the kidney, but the basal rate of NO synthesis appears to be different in different vascular beds. In fact, in our study the systemic infusion of L-NAME induced a variable degree of blood flow reduction in the three arteries (29% RBF, 216% MBF, and 219% IBF during PLN in Group B). It is then conceivable that the stimulation of NO synthesis may not uniformly affect vascular resistances in all vascular beds. A second possibility is the existence of local differences in vascular reactivity to vasoconstrictor stimuli. Insulin has been reported to enhance the expression and circulating levels of endothelin-1.36 An elevation in plasma norepinephrine concentrations in response to acute hyperinsulinemia was described,1 and subsequently confirmed by other authors with even lower doses of insulin.3,21 In addition, insulin has been reported to cause increased cardiovascular reactivity to the pressor effects of norepinephrine,37 although infusion of norepinephrine during a hyperinsulinemic state failed to cause any alteration in human forearm blood flow.28 These controversial data may reinforce the existence of vascular heterogeneity in the number or affinity of receptors for vasoconstrictor factors. A final possibility consists of the combination of the two previous mechanisms. A specific balance between insulin-induced vasodilation and vasoconstriction would determine net hemodynamic outcome in each vascular bed. The last aim of our study was to investigate the effects of physiologic hyperinsulinemia on blood pressure. In our model, hyperinsulinemia per se did not induce any significant change in MAP, in agreement with the large body of previous evidence in humans and dogs.3–7,27,28,37 However, when partial NO inhibition preceded and paralleled the same level of hyperinsulinemia, a significant rise in MAP was observed. In this line, NO blockade has been recently reported in humans to impair insulin-mediated skeletal muscle vasodilation, associated with an increase in mean arterial pressure, during an euglycemic hyperinsulinemic clamp.28 These data suggest the possibility that physiologic hyperinsulinemia may be a pressor mechanism in situations of endothelial derangement related to a defective NO system. This hypothesis might rec-
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oncile the controversy between lack of effect of acute and short-term hyperinsulinemism in the genesis of hypertension,3–7,38 and the epidemiologic correlation between hypertension, hyperinsulinemia, and insulin resistance.39,40
16.
Creager MS, Liang CS, Coffman JD: Beta adrenergic mediated vasodilator response to insulin in the human forearm. J Pharmacol Exper Ther 1985;235:709 –714.
17.
Gelfand RA, Barret EJ: Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest 1987;80:1– 6.
ACKNOWLEDGMENTS
18.
Baron AD, Brechtel-Hook G, Johnson A, et al: Skeletal muscle blood flow. A possible link between insulin resistance and blood pressure. Hypertension 1993;21: 129 –135.
19.
Jamerson KA, Julius S, Gudbrandsson O, et al: Reflex sympathetic activation induces acute insulin resistance in the human forearm. Hypertension 1993;21:618 – 623.
In memoriam Mary Olsen. We are indebted to Rodney Bolterman for excellent technical assistance.
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