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(Hypertension. 1995;25:994-1002.)
© 1995 American Heart Association, Inc.

Articles

Hemodynamic and Renal Responses to Chronic Hyperinsulinemia in Obese, Insulin-Resistant Dogs

John E. Hall; Michael W. Brands; Dion H. Zappe; William N. Dixon; H. Leland Mizelle; Glenn A. Reinhart; Drew A. Hildebrandt

From the Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson.

	Abstract

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Abstract We previously reported that chronic hyperinsulinemia does not cause hypertension in normal insulin-sensitive dogs. However, resistance to the metabolic and vasodilator effects of insulin may be a prerequisite for hyperinsulinemia to elevate blood pressure. The present study tested this hypothesis by comparing the control of systemic hemodynamics and renal function during chronic hyperinsulinemia in instrumented normal conscious dogs (n=6) and in dogs made obese and insulin resistant by feeding them a high-fat diet for 6 weeks (n=6). After 6 weeks of the high-fat diet, body weight increased from 24.0±1.2 to 40.9±1.2 kg, arterial pressure rose from 83±5 to 106±4 mm Hg, and cardiac output rose from 2.98±0.29 to 5.27±0.54 L/min. Insulin sensitivity, assessed by fasting hyperinsulinemia and by the hyperinsulinemic euglycemic clamp technique, was markedly reduced in obese dogs. Insulin infusion (1.0 mU/kg per minute for 7 days) in obese dogs elevated plasma insulin from 42±12 µU/mL to 95 to 219 µU/mL but failed to increase arterial pressure, which averaged 106±4 mm Hg during control and 102±4 mm Hg during 7 days of insulin infusion. Hyperinsulinemia for 7 days in obese dogs elevated heart rate from 116±8 to 135±7 beats per minute but caused no significant changes in cardiac output, in contrast to normal dogs (n=6), in which marked increases in cardiac output (31±5% after 7 days) and decreases in total peripheral resistance occurred during chronic insulin infusion. Thus, chronic hyperinsulinemia did not raise blood pressure in obese dogs even though they were resistant to the metabolic and vasodilator effects of insulin. These observations provide no evidence that hyperinsulinemia causes hypertension, even in the presence of insulin resistance, in obese dogs.

Key Words: obesity • insulin • cardiac output • vasodilation • heart rate • kidney • blood flow

	Introduction

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The possibility that insulin resistance and hyperinsulinemia may contribute to human essential hypertension, especially when associated with obesity, has generated considerable interest. Epidemiological studies have supported this hypothesis by demonstrating that obese, hypertensive subjects tend to be hyperinsulinemic and insulin resistant when compared with lean, normotensive subjects.^{1 2 3 4} Moreover, some studies have shown a correlation between blood pressure (BP) and plasma insulin concentration in lean as well as obese hypertensive subjects.⁵ Additional support for this concept comes from short-term studies demonstrating that insulin has multiple effects on the kidney, the sympathetic nervous system, and the cardiovascular system that, if sustained, could raise BP.^{6 7 8 9} However, other studies have failed to find a correlation between insulin and BP,^{10 11 12 13} and it is now recognized that insulin has pressor as well as depressor (eg, vasodilator) effects on the cardiovascular system that could elevate or reduce BP, depending on their relative importance.^{14 15} Thus, the effects of insulin on BP are complex, and there is considerable controversy about whether insulin is capable of exerting sustained effects on cardiovascular and renal function that are necessary to cause chronic hypertension.

We previously reported that in normal dogs chronic hyperinsulinemia did not raise BP or potentiate the hypertensive effect of other pressor systems, such as the renin-angiotensin and adrenergic systems.^{16 17 18} In fact, insulin infusion lowered BP and caused marked peripheral vasodilation in normal dogs.¹⁸ Similar results have been reported during acute insulin infusion in normotensive, lean humans.^{9 19 20} However, additional abnormalities associated with obesity besides hyperinsulin- emia per se may be essential for insulin to cause hypertension. One factor that has been postulated to contribute to the hypertensive effects of insulin is resistance to the metabolic and vasodilator effects of insulin.^{15 19} In normal dogs that are sensitive to the metabolic and vasodilator effects of insulin, insulin infusion may lead to a decrease in peripheral vascular resistance that offsets any hypertensive effects caused by sodium retention or activation of the sympathetic nervous system. In our previous studies,¹⁸ hyperinsulinemia for 7 days in normal dogs reduced total peripheral resistance (TPR) by more than 30% and decreased mean arterial pressure (MAP) while markedly increasing cardiac output. Acute insulin infusion in normotensive humans and in patients with borderline hypertension also reduced forearm vascular resistance and increased blood flow.^{9 19} Recent studies suggest that the short-term vasodilator response to hyperinsulinemia may be attenuated in obese, insulin-resistant subjects.²⁰ However, no studies have determined whether the chronic vasodilator effects of insulin are blunted in obese, insulin-resistant subjects.

Based on results of short-term studies, hyperinsulin- emia has been postulated to increase BP only when insulin resistance is present and the peripheral vasodilator effects of insulin are impaired.^{15 19} We designed the present study to test this hypothesis by determining whether obese dogs are resistant to the long-term vasodilator effects of insulin and whether chronic hyperinsulinemia raises BP in the presence of insulin resistance. In addition, our studies characterized systemic hemodynamics, renal function, and endocrine changes associated with chronic hyperinsulinemia in obese, insulin-resistant dogs.

	Methods

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Experiments were conducted in 12 chronically instrumented mongrel dogs that were conditioned before study. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and were carried out according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and according to the guidelines of the Animal Welfare Act.

Surgical procedures were conducted under aseptic conditions and pentobarbital sodium anesthesia (30 mg/kg). Catheters (Tygon, Norton Plastics Synthetics Division) were implanted in the femoral arteries and veins for measurement of arterial pressure and venous infusions. The catheters were tunneled subcutaneously, exteriorized in the scapular region for protection, and filled with heparin solution (1000 USP U/mL). An electromagnetic flow probe (Zepeda Instruments) was implanted around the ascending aorta through an incision at the fourth intercostal space, and the leads were exteriorized in the scapular region. The dogs were permitted to recover from surgery, antibiotics were administered daily, and rectal temperatures were monitored to ensure that the dogs were afebrile throughout the studies.

After recovery, the dogs were placed in individual metabolic cages in a quiet air-conditioned room with a 12-hour light/dark cycle and fitted with harnesses containing a pressure transducer (Cobe) mounted at heart level. One of the femoral artery catheters was connected to the pressure transducer so that MAP could be recorded continuously on a polygraph (model 7D, Grass Instruments). MAP signals from the Grass recorder were sent to an analog-digital convertor and analyzed with a digital computer (TurboX-T, PCs Limited) using software developed in our laboratory. Analog signals from the polygraph were sampled in bursts of 12 seconds each minute, 24 hours a day, and the digitized data were processed on the computer to determine systolic and diastolic pressures, MAP, and heart rate. Cardiac output signals were monitored with an electromagnetic flowmeter (Zepeda Instruments) throughout the day. Analog signals from the flowmeter were sampled in bursts of 12 seconds each minute, 24 hours a day, along with the arterial pressure signals. Aortic flow signals were processed on the computer for determination of stroke volume, cardiac output, peak aortic flow, and baseline diastolic flow. By comparing the diastolic flow baseline with the previously calibrated zero-voltage baseline, baseline drift of the flow probe could be corrected on a beat-by-beat basis to allow precise determinations of cardiac output. TPR was computed on a beat-by-beat basis using the cardiac output and arterial pressure signals. The MAP, heart rate, cardiac output, and TPR for each day were calculated from values recorded over an 18-hour period, between 2 PM and 8 AM. All routine care of the dogs, including feeding and cleaning of the cages, studies of renal function, and blood sampling, were done between 8 AM and 2 PM.

For continuous infusion of various solutions, one of the femoral venous catheters was connected to a roller infusion pump (model 375A, Sage Instruments). All solutions were pumped through a disposable, sterile filter (Cathivex, Millipore) to prevent contaminants and bacteria from passing into the venous infusion catheters. The infusion tubing and cables from the pressure transducers were protected by a flexible vacuum hose attached to a harness that permitted the dogs to move freely in the cages.

The dogs were fed two cans (447 g per can) per day of a sodium-deficient diet (H/D, Hill's Pet Products) that provided approximately 7 mmol sodium and 65 mmol potassium per day throughout the study. They were also given 5 mL of a vitamin syrup (VAL Syrup, Ft Dodge Laboratories) each day. Total sodium intake, including the food and the intravenous infusion of sodium chloride, was held constant throughout the study at approximately 80 mmol/d.

Experimental Protocols
After the dogs were placed in metabolic cages and all infusions were started, 10 to 14 days were allowed for the dogs to achieve sodium balance and for the acquisition of stable control measurements. During that time, the dogs were trained to lie quietly while studies of renal function were conducted and blood samples obtained. After at least 5 days of stable control measurements, cooked beef fat (0.5 to 0.9 kg) was added to the regular diet of one group of dogs (n=6). The high-fat diet was maintained for 6 weeks, and measurements of systemic hemodynamics and renal function were conducted.

After 6 weeks of the high-fat diet, 45 mL/d of sterile water was infused with a syringe pump (Harvard Apparatus), and approximately 750 to 1070 mL/d of sterile water was infused intravenously with a roller pump to provide the vehicles for the insulin and glucose infusions during the experimental period. After 7 to 10 days of control measurements, an intravenous infusion of insulin was started at a rate of 1.0 mU/kg per minute and continued for 7 days. Plasma glucose concentration was held relatively constant using a glucose clamp procedure in which a 50% solution of glucose was infused along with the insulin. The rate of glucose infusion required to maintain plasma concentration constant was calculated with a mathematical model of glucose and insulin kinetics.¹⁶ During the first day of insulin infusion, glucose measurements were made with a blood glucose monitor (Accu-Chek II, Boehringer Mannheim) to ensure that hypoglycemia did not occur. Thereafter, quantitative assessments of plasma glucose were made under fasting conditions, as described below. No attempt was made to precisely regulate blood glucose concentration after feeding. Instead, the rate of glucose infusion selected for each dog was held constant during chronic hyperinsulinemia. After 7 days of insulin and glucose infusions, postcontrol measurements were made for at least 5 more days. The high-fat diet was continued during the insulin infusion and postcontrol periods.

The same protocol for chronic insulin infusion was followed in a second group of lean dogs (n=6). These dogs were fed the same diet as described above except that beef fat was not added to the food. Sodium intake was also fixed at approximately 80 mmol/d, as described above. After 7 to 10 days of control measurements, insulin was infused intravenously at a rate of 1.0 mU/kg per minute for 7 days, and plasma glucose concentration was held relatively constant with the glucose clamp method.

Analytical Methods
Glomerular filtration rate (GFR) and effective renal plasma flow were determined from total clearances of [¹²⁵I]iothalamate (Glofil, Isotex Diagnostics) and [¹³¹I]iodohippurate (Hippuran, ER Squibb & Sons, Inc), respectively, as previously described.²¹ Distribution space of [¹²⁵I]iothalamate was used as an index of extracellular fluid volume. Plasma and urinary sodium and potassium concentrations were determined with ion-specific electrodes (Nova Biomedical). Plasma and urinary chloride concentrations were made by coulometric titration with a chloridometer (Haake Buchler). Plasma protein concentration was measured by refractometry (TS Meter, American Optical), and plasma glucose concentration was determined by the hexokinase method (Sigma Diagnostics).

Plasma renin activity (PRA) was measured by radioimmunoassay using ¹²⁵I–angiotensin I (Ang I) from New England Nuclear and antibody from Chemicon. Aldosterone was extracted from plasma with 7 vol dichloromethane, and the dried extract was reconstituted with phosphate gelatin buffer and measured by radioimmunoassay using ¹²⁵I-aldosterone from Amersham and liquid phase antibody (Diagnostic Products Corp). Plasma insulin concentration was measured by radioimmunoassay (Diagnostic Products). Plasma epinephrine and norepinephrine concentrations were measured by high-performance liquid chromatography with electrochemical detection. Before assay, catecholamines were extracted by adsorption on alumina and eluted with 0.1N perchloric acid.²²

Statistical Analyses
Experimental data were compared with control data by ANOVA and, when appropriate, with Dunnett's t test for multiple comparisons.^{23 24} Statistical significance was considered to be at a value of P<.05. All data are expressed as mean±SEM, unless otherwise indicated.

	Results

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Effects of 6 Weeks of a High-Fat Diet
Table 1 shows the effects of 6 weeks of a high-fat diet on cardiovascular, renal, and endocrine functions. Body weight increased by approximately 16.9 kg, whereas MAP, cardiac output, and heart rate increased by 28%, 77%, and 68%, respectively. TPR decreased by approximately 31%; however, when indexed for body weight, TPR index increased by 26% and cardiac index did not change significantly after 6 weeks of the high-fat diet. The high-fat diet was also associated with marked increases in GFR (61%), effective renal plasma flow (86%), and sodium iothalamate space (44%), an index of extracellular fluid volume. PRA almost doubled, and plasma aldosterone concentration tended to increase although the change was not significant after 6 weeks of the high-fat diet. Fasting plasma insulin concentration increased from 12.2±2.8 to 42.3±11.9 µU/mL, but plasma glucose concentration did not change significantly. The high-fat diet was also associated with significant increases in plasma cholesterol, norepinephrine, and protein concentrations. There were no significant changes in plasma sodium or chloride concentrations, but plasma potassium concentration decreased slightly after 6 weeks of the high-fat diet.

View this table: [in this window] [in a new window]   Table 1. Baseline Values in Conscious Dogs Before and After 6 Weeks of a High-Fat Diet

Chronic Insulin Infusion in Obese Dogs
After 6 weeks of the high-fat diet, insulin was infused intravenously for 7 days at a rate of 1.0 mU/kg per minute while plasma glucose concentration was maintained relatively constant by simultaneous glucose infusion. The glucose infusion rate needed to maintain euglycemia averaged 9.6 mg/kg per minute in obese dogs, compared with 14 mg/kg per minute in lean dogs, even though plasma insulin concentration was significantly higher in obese than in normal dogs during 7 days of insulin infusion. Thus, the sensitivity of obese dogs to the effects of insulin on glucose disposal was markedly reduced compared with lean dogs.

As shown in Table 2, insulin infusion raised plasma insulin concentration approximately fivefold during the first day, and after 3 and 6 days plasma insulin concentration averaged 150 and 95 µU/mL, compared with a control value of 42±12 µU/mL. Plasma glucose concentration increased slightly on the first day of insulin infusion but thereafter was not significantly different from control. PRA increased from 0.92±0.21 to 2.29±0.38 ng Ang I/mL per hour after 6 days of insulin infusion. Plasma aldosterone concentration also tended to increase, but the changes were statistically significant only on day 3 of insulin infusion. Plasma potassium concentration decreased significantly during 7 days of insulin infusion. There were no significant changes in plasma sodium or chloride concentrations, but plasma protein concentration decreased from a control level of 7.76±0.19 to 6.79±0.23 g/100 mL after 6 days of insulin infusion. Hematocrit and plasma epinephrine and norepinephrine concentrations during insulin infusion in obese dogs did not change significantly.

View this table: [in this window] [in a new window]   Table 2. Effects of Insulin Infusion in Obese Insulin-Resistant Dogs

Fig 1 shows the effects of 7 days of insulin infusion on heart rate, cardiac output, and TPR. Although the high-fat diet markedly elevated heart rate, insulin infusion for 7 days further increased heart rate from 116±8 beats per minute (bpm) to an average of 135±7 bpm. Insulin infusion did not significantly alter cardiac output, which averaged 5.27±0.54 L/min during control, 5.92±0.59 L/min during 7 days of insulin infusion, and 5.82±0.59 L/min during the 7-day postcontrol period after the insulin infusion was stopped. TPR decreased slightly but significantly during insulin infusion, averaging 20.5±1.7 mm Hg/L per minute during control and 17.7±1.3 mm Hg/L per minute during 7 days of insulin infusion; however, this small decrease in TPR may not have been directly related to insulin infusion because TPR remained reduced during the postcontrol period, averaging 18.1±1.3 mm Hg/L per minute during the 7 postcontrol days, a value that was not significantly different from TPR during 7 days of insulin infusion. The trend toward a small increase in cardiac output and decrease in TPR may be related to the 3.1±0.3 kg weight gain that occurred between the control and postinsulin infusion periods as the dogs remained on the high-fat diet.

View larger version (47K): [in this window] [in a new window]   Figure 1. Bar graphs show effect of insulin infusion (1.0 mU/kg per minute IV) for 7 days in obese dogs on heart rate, cardiac output, and total peripheral resistance.

Despite the attenuated decrease in TPR, insulin infusion did not significantly alter MAP in obese dogs; during 7 days of insulin infusion, MAP averaged 102±4 mm Hg, compared with 106±4 mm Hg during the control and 102±4 mm Hg during the postcontrol periods (Fig 2).

View larger version (41K): [in this window] [in a new window]   Figure 2. Bar graph shows effect of insulin infusion (1.0 mU/kg per minute IV) for 7 days in obese dogs on mean arterial pressure.

Insulin infusion in obese dogs caused a modest decrease in urine volume (Fig 3), from a control value of 1400±129 to 813±26 mL/d on the first day; on subsequent days, urine volume gradually returned toward control. However, because water drinking increased from 446±154 mL/d to an average of 865±285 mL/d, there was net fluid retention during 7 days of insulin infusion in obese dogs. Insulin tended to decrease urinary sodium excretion, which averaged 54.3±4.7 mmol/d during control and 46.0±3.9 mmol/d during 7 days of insulin infusion, although the changes were not statistically significant. Urinary potassium excretion decreased significantly during the first 3 days of insulin infusion and then returned toward control; for the entire 7 days of insulin infusion, urinary potassium excretion averaged 49.3±2.8 mmol/d, compared with 59.6±5.2 mmol/d during control.

View larger version (44K): [in this window] [in a new window]   Figure 3. Bar graphs show effect of insulin infusion (1.0 mU/kg per minute IV) for 7 days in obese dogs on urine volume, urinary sodium excretion, and urinary potassium excretion.

The fluid and potassium retention caused by hyperinsulinemia in obese dogs was not caused by decreased GFR, which averaged 104±6% of control during insulin infusion (Fig 4). There were also no significant changes in effective renal plasma flow or renal vascular resistance during 7 days of hyperinsulinemia in obese dogs.

View larger version (36K): [in this window] [in a new window]   Figure 4. Bar graphs show effect of insulin infusion (1.0 mU/kg per minute IV) for 7 days in obese dogs on glomerular filtration rate, effective renal plasma flow, and renal vascular resistance.

Chronic Insulin Infusion in Normal Dogs
Insulin infusion in normal dogs raised plasma insulin concentration by approximately fourfold to fivefold during the first day, and after 6 days, plasma insulin concentration averaged 105.5±14.4 µU/mL, compared with a control value of 22.1±1.5 µU/mL (Table 3). Plasma glucose concentration remained relatively constant throughout the 7 days of insulin infusion, averaging 108±4 g/100 mL during control and 93±7 g/100 mL during 7 days of hyperinsulinemia. PRA increased from 0.57±0.16 to 2.25±0.47 ng Ang I/mL per hour after 6 days of insulin infusion. Plasma potassium concentration decreased significantly on days 1 and 3 of insulin infusion but returned toward control after 6 days. Plasma sodium and chloride concentrations did not change significantly during 7 days of insulin infusion, but plasma protein concentration decreased from a control level of 6.78±0.36 to 5.76±0.46 g/100 mL after 6 days. Hematocrit did not change significantly during insulin infusion in normal dogs.

View this table: [in this window] [in a new window]   Table 3. Effects of Insulin Infusion in Normal Dogs

Insulin infusion for 7 days in normal dogs increased heart rate from 69±2 bpm to an average of 88±2 bpm (Table 4). Cardiac output increased gradually to 131±5% of control after 7 days, and TPR decreased to 76±4% of control. MAP did not change significantly, averaging 87±5 mm Hg during control and 89±5 mm Hg during 7 days of insulin infusion.

View this table: [in this window] [in a new window]   Table 4. Effects of Insulin Infusion on Hemodynamics and Renal Function in Normal Dogs

Insulin infusion in normal dogs caused significant retention of sodium, potassium, and water. Urine volume decreased from a control value of 1828±73 mL/d to 1248 and 1090 mL/d on the first 2 days of insulin infusion; thereafter, urine volume returned toward control, despite significant increases in water drinking, which averaged 68±14 mL/d during control and 207±61 mL/d during 7 days of insulin infusion. Thus, insulin infusion in normal dogs caused a substantial increase in cumulative water balance. Urinary sodium excretion decreased significantly during the first 2 days of insulin infusion and then returned toward control. Potassium excretion also decreased significantly during the first 4 days of insulin infusion and then returned toward control; for the entire 7 days of insulin infusion, urinary potassium excretion averaged 42.4±3.3 mmol/d compared with a control value of 55.6±1.5 mmol/d.

In normal dogs, insulin significantly increased GFR to 124±6% of control after 6 days of infusion. Effective renal plasma flow averaged 117±8% of control after 6 days of insulin infusion. Thus, the sodium and water retention during insulin infusion was not due to decreased GFR or renal vasoconstriction. Sodium iothalamate space, an index of extracellular fluid volume, increased to 119±4% of control after 6 days of insulin infusion.

	Discussion

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An important finding of this study is that 7 days of hyperinsulinemia did not elevate arterial pressure in normal dogs or in obese dogs that were resistant to the metabolic and vasodilator actions of insulin. In normal dogs, 7 days of hyperinsulinemia caused marked peripheral vasodilation and increased cardiac output. In contrast, cardiac output did not increase in obese dogs during hyperinsulinemia, and the long-term peripheral vasodilator effects of insulin were greatly attenuated or abolished. These observations suggest that the presence or absence of insulin resistance and/or peripheral vasodilation does not markedly alter the BP responses to 7 days of hyperinsulinemia in dogs.

Previous reports of correlations between insulin resistance, hyperinsulinemia, and hypertension, especially in obese subjects, have led to the hypothesis that hyperinsulinemia may be an important cause of hypertension.^{25 26 27 28} Indirect support for this concept has come from multiple short-term studies demonstrating that insulin has renal and sympathetic nervous system effects that could lead to chronic hypertension if they were sustained.^{6 7} As a test of this hypothesis, we previously studied the effects of chronic hyperinsulinemia, comparable to that found in obese subjects, on BP regulation.^{16 17 18} Our results indicated that insulin infusion for as long as 28 days did not elevate BP in normal dogs, even when kidney mass was reduced and when dogs were maintained on a high sodium intake. In addition, hyperinsulinemia did not potentiate the long-term hypertensive effects of other pressor hormones, such as Ang II and norepinephrine.¹⁶ Short-term studies in humans have also failed to find a hypertensive effect of insulin,^{9 19 20} and patients with very high plasma insulin concentrations caused by insulinoma are not hypertensive.^{29 30} Thus, chronic hyperinsulinemia per se has not been demonstrated to elicit significant BP increases in dogs or humans, although we have previously reported a hypertensive effect of insulin in rats.^{31 32}

The failure of acute or chronic hyperinsulinemia to elevate BP in normotensive, insulin-sensitive subjects has been suggested to result from offsetting effects of a peripheral vasodilator action of insulin that tends to lower BP and various hypertensive effects of insulin that elevate BP.^{15 19} Moreover, impaired peripheral vasodilation has been postulated to unmask a hypertensive effect of insulin in obese, insulin-resistant subjects. Support for this hypothesis has come from short-term studies demonstrating that insulin-induced vasodilation is attenuated in obese, insulin-resistant subjects.^{19 20 33} However, no previous studies, to our knowledge, have determined whether the long-term vasodilator action of insulin is blunted in insulin-resistant subjects or whether chronic hyperinsulinemia would raise BP in obese, insulin-resistant subjects. Results of the present study indicate that the chronic vasodilator action of insulin is markedly attenuated in obese, insulin-resistant dogs, but despite this resistance to the vascular effects of insulin, hyperinsulinemia for 7 days does not raise BP.

Hemodynamic Actions of Insulin in Obese Dogs
The mechanisms by which obesity attenuates insulin-induced vasodilation are unclear but could be related to the generalized resistance to the effects of insulin on tissue metabolism. In insulin-sensitive subjects, acute hyperinsulinemia stimulates skeletal muscle glucose uptake and increased oxygen consumption,^{19 20} which in turn could activate local vasodilator mechanisms that increase tissue blood flow. To the extent that the vasodilator action of insulin is mediated through local metabolic mechanisms, resistance to the effects of insulin on glucose uptake would attenuate increases in tissue blood flow and cardiac output during chronic hyperinsulinemia.

In addition to its metabolic actions, insulin may also induce vasodilation by ß-adrenergic mechanisms,³⁴ by increasing release of endothelium-derived relaxing factors³⁵ or by altering intracellular calcium metabolism via direct effects on membrane calcium transport.³⁶ Recent studies suggest that hyperinsulinemic, insulin-resistant rats have increased vascular reactivity, reduced membrane calcium-ATPase activity, and impaired vascular smooth muscle calcium efflux compared with controls.³⁶ These changes could raise intracellular calcium levels, thereby diminishing the vasodilator action of insulin.³⁶ We did not design the present study to test the importance of these different actions, and it is possible that the mechanisms responsible for the long-term vasodilator effects of insulin may be quantitatively different from those that elicit the short-term effects of insulin. However, our results indicate that obesity impairs the long-term vasodilator action of insulin in association with a resistance to its metabolic effects. Further studies are needed to determine the specific mechanisms linking insulin resistance with attenuation of insulin-induced vasodilation.

Effects of Insulin on Heart Rate in Obese Dogs
Another consistent cardiovascular effect of insulin is increased heart rate. In normal dogs, 7 days of hyperinsulinemia increased heart rate by approximately 30% to 35%, similar to results from our previous studies.^{17 18} Short-term studies in humans have also shown that hyperinsulinemia causes tachycardia, even when euglycemia is maintained.⁹ Despite the presence of insulin resistance and impaired peripheral vasodilator responses to insulin, obese dogs also demonstrated significant increases in heart rate during 7 days of hyperinsulinemia. This tachycardia occurred even though control heart rates in obese dogs were already elevated by approximately 68% after 6 weeks of the high-fat diet. Thus, obesity and insulin resistance do not abolish the tachycardia associated with chronic hyperinsulinemia.

The mechanisms responsible for the increased heart rate associated with chronic hyperinsulinemia have not been fully elucidated, but our recent preliminary studies indicate that this response is not abolished by ß-adrenergic blockade.³⁷ Therefore, the increase in heart rate associated with chronic hyperinsulinemia is probably not entirely due to increased sympathetic activity. It is possible that the tachycardia associated with chronic hyperinsulinemia may be mainly due to withdrawal of parasympathetic tone, similar to the parasympathetic-mediated increase in heart rate associated with obesity.³⁸ However, further studies are needed to elucidate the precise mechanisms by which hyperinsulinemia influences heart rate regulation.

Renal Actions of Insulin in Obese Dogs
In the present study, chronic hyperinsulinemia transiently decreased renal excretion of sodium, water, and potassium in normal dogs. Previous short-term studies in animals and humans have demonstrated the antinatriuretic effects of insulin.^{6 8} Moreover, the short-term sodium-retaining effects of insulin appear to be preserved in obese, insulin-resistant humans.³⁹ Results from the present study indicate that the long-term effects of insulin on renal water and potassium excretion are also maintained in obese, insulin-resistant dogs; however, the sodium-retaining effects of insulin appeared to be attenuated in obese compared with normal dogs. The reason for the blunted sodium-retaining effects of insulin in obese dogs is unclear. Previous studies have suggested that an important part of the antinatriuretic effect of hyperinsulinemia may be due to indirect effects, such as hypokalemia or decreased arterial pressure.⁴⁰ However, insulin infusion caused no significant changes in MAP in obese or normal dogs, and the degree of hypokalemia caused by insulin infusion was similar in normal and obese dogs. One notable difference between normal and obese dogs is that insulin caused marked peripheral vasodilation in normal dogs, with little or no change in peripheral vascular resistance in obese dogs. Therefore, it is possible that some of the sodium and water retention associated with hyperinsulinemia in normal dogs may have been secondary to peripheral vasodilation and/or increased peripheral vascular capacity. However, we did not design the present study to examine the mechanisms by which obesity alters the renal excretory responses to hyperinsulinemia.

The mechanisms for insulin-induced volume retention in the present study are not entirely clear but are not related to renal vasoconstriction or decreased GFR. In fact, during the first 3 days of insulin infusion, when fluid retention was most apparent, there was actually a slight (albeit statistically insignificant) increase in GFR. Thus, the long-term antidiuretic effect of insulin appears to be mediated by increased tubular reabsorption. This conclusion is in agreement with previous short-term studies which have shown that insulin increases renal sodium and water absorption.⁸ The tubular site at which insulin increases reabsorption was not determined in the present study, but previous short-term experiments in rats suggest that insulin may stimulate reabsorption mainly in the loop of Henle.⁸

After several days of insulin infusion, there was an escape from fluid retention and a return of urinary excretion toward normal. The mechanisms responsible for renal escape are not related to increased BP, because insulin infusion in obese dogs tended to lower BP slightly. Decreased formation of Ang II and aldosterone cannot explain the escape from sodium and water retention, because PRA and aldosterone concentration tended to increase during insulin infusion. In fact, increases in Ang II could contribute to the sodium and water retention associated with hyperinsulinemia. One possible mechanism that could offset the effect of insulin on tubular reabsorption is the tendency toward increased GFR and filtered sodium load, which may be a compensatory response to increased loop of Henle sodium chloride reabsorption. For example, increased loop of Henle sodium chloride reabsorption would tend to decrease macula densa sodium chloride delivery, which could in turn cause a macula densa feedback-mediated renal vasodilation.^{14 41} However, this hypothesis must be considered speculative until it can be tested with experimental studies.

The reductions in urinary potassium excretion in obese dogs were even greater than the decreased sodium excretion observed during chronic hyperinsulinemia. Previous studies have suggested that hyperinsulinemia may increase potassium uptake in extrarenal tissues, causing hypokalemia.⁴² In the present study, chronic hyperinsulinemia induced a modest hypokalemia in obese, insulin-resistant dogs similar to that observed in normal dogs. Thus, the effects of insulin on potassium metabolism appeared to be similar in obese, insulin-resistant and normal dogs. Although the mechanisms responsible for the antikaliuresis were not the main focus of the present study, a reduction in distal sodium chloride delivery caused by increased loop of Henle reabsorption would tend to reduce potassium excretion.

Effects of High-Fat Diet on Cardiovascular and Renal Functions
Previous studies by Rocchini et al⁴³ and studies in our laboratory⁴⁴ have detailed the cardiovascular, renal, and endocrine responses to a high-fat diet in dogs. This model of obesity mimics obesity in humans in many ways. For example, as demonstrated in the present study and in previous studies with this model,^{43 44} a high-fat diet raises arterial pressure, cardiac output, heart rate, PRA, and plasma insulin concentration and causes marked sodium retention mainly by increasing renal tubular sodium reabsorption. The mechanisms that initiate these changes, however, have not been fully elucidated.

A question that has stirred considerable controversy is whether hyperinsulinemia contributes to the changes in cardiovascular and renal function associated with obesity. Although our studies were not designed specifically to test this hypothesis, they provide insight into the cardiovascular renal actions that chronic hyperinsulin- emia is capable of causing. Even though plasma insulin concentrations were elevated by approximately threefold to fourfold after 6 weeks of the high-fat diet, 7 days of insulin infusion to further raise plasma insulin concentration caused additional increases in heart rate and PRA and further water and potassium retention, indicating that any possible contribution of insulin to changes in these variables in obesity was not maximal. However, hyperinsulinemia caused no significant changes in MAP in either obese or lean dogs.

One could argue that increased insulin levels may activate pressor mechanisms in obesity that are maximally engaged before infusion of exogenous insulin; if this were the case, additional hyperinsulinemia caused by insulin infusion would cause no additional increases in arterial pressure. Although our results cannot rule out this possibility, it is important to note that hyperinsulin- emia per se has not been found to elevate BP in nonobese or obese humans or dogs.^{45 46} Also, most previous studies indicate that the metabolic effects of insulin and its effects on the kidney and sympathetic nervous system, which have been postulated to mediate the BP actions of insulin, are not yet maximal at a concentration of 40 to 50 µU/mL,^{45 46 47} the average plasma concentration of insulin in obese dogs before insulin infusion. Also, a further short-term elevation of plasma insulin concentration causes additional increases in muscle sympathetic activity in obese humans, with no change in arterial pressure,⁴⁷ similar to the results obtained in the present study.

The results of the present study provided a specific test of the hypothesis that insulin may cause hypertension under conditions in which the vasodilator action of insulin is attenuated. Our results indicate that 7 days of hyperinsulinemia did not elevate BP in insulin-resistant dogs in which the vasodilator action of insulin was almost completely abolished. However, additional studies are needed to determine whether blockade of endog- enous insulin secretion has any significant long-term effect on BP regulation in obese, insulin-resistant subjects.

In summary, our results indicate that obesity, caused by feeding dogs a high-fat diet, is associated with resistance to the metabolic effects of insulin as well as a marked impairment of the long-term vasodilator action of insulin. The renal effects of insulin, including increased tubular reabsorption and renal vasodilation as well as the effect of insulin to raise heart rate, appear to be preserved in obese, insulin-resistant dogs. However, chronic hyperinsulinemia did not elevate BP even in the presence of insulin resistance and impaired peripheral vasodilation. These results provide no evidence that hyperinsulinemia causes hypertension in the presence of insulin resistance in obese dogs.

	Acknowledgments

 
This research was supported by grants HL-51971, HL-39399, and HL-23502 from the National Institutes of Health, Bethesda, Md. The authors thank Ivadelle Heidke and Susie Zuller for excellent secretarial assistance. We also thank Cathy Garrity, Calvin Torrey, and Christine Mitchell for technical assistance.

	Footnotes

 
Reprint requests to John E. Hall, PhD, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216-4505. E-mail JEH@fiona.usmed.edu.

Received October 27, 1994; first decision November 29, 1994; accepted January 11, 1995.

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