Chronic

hyperinsulinemia

and blood pressure regulation

JOHN E. HALL, THOMAS G. COLEMAN, H. LELAND MIZELLE, AND MANIS J. SMITH, JR. (With the Technical Assistance of Robert W. Seaton) Department of Physiology and Biophysics, University of Mississippi Medical Center, e Jackson, Mississippi 39216

HALL, JOHN E., THOMAS G. COLEMAN, H. LELAND MIZELLE, AND MANIS J. SMITH, JR. Chronic hyperinsulinemia and Hood pressure regulation. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F722-F731, 1990.-The aims of this study were to determine whether chronic hyperinsulinemia, comparable to that found in obese hypertensives, elevates mean arterial pressure (MAP) or potentiates the hypertensive effects of angictensin II (ANG II). Studies were conducted in conscious dogs with kidney mass reduced by 70% in order to increase their susceptibility to hypertensive stimuli. Insulin infusion (0.5 or 1.0 mu. kg-l. min iv) for 7 days with plasma glucose held constant raised plasma insulin more than fivefold but did not increase MAP in four dogs on 138 meq/day Na intake. In seven dogs maintained on a high Na intake (319 meq/day), insulin infusion (1.0 mu. kg-l. min) for 28 days raised fasting insulin from 9.8 t 1.5 to 56-78 @U/ml but did not increase MAP, which averaged 106 t 2 mmHg during control and 102 t 2 mmHg during 28 days of insulin infusion. Insulin caused transient sodium and potassium retention followed by renal “escape” that was associated with increased glomerular filtration rate (12-27%). Plasma renin activity and plasma aldosterone were not altered by insulin. In five dogs infused with ANG II (2.0 ng kg-‘. min-‘) to cause mild hypertension, insulin infusion (1.0 mu. kg-‘. min-‘) for 6-28 days did not increase MAP further. Thus chronic hyperinsulinemia did not elevate MAP, even when kidney mass was reduced, and did not potentiate the hypertensive effects of ANG II. These findings suggest that additional factors besides hyperinsulinemia per se are responsible for obesity-associated hypertension. l

insulin; hypertension; filtration; angiotensin

obesity;

sodium

excretion;

glomerular

OF HYPERTENSION is severalfold greater in obese than in nonobese individuals and an important correlation between body weight and blood pressure has been previously noted (11,29). However, the mechanisms responsible for the link between obesity and hypertension remain obscure. The possibility that hyperinsulinemia might play a role in obesity-associated hypertension has been suggested by several investigators (4, 6, 8, 23). Hyperinsulinemia is a common feature of obese patients with hypertension and there is a significant correlation between blood pressure and plasma insulin concentration in these individuals (6, 23). When obese subjects are placed on a low-calorie diet, plasma insulin levels and blood pressure often decrease in parallel even when sodium intake is maintained relatively constant (28, 34). Berglund et al. (4) observed that untreated hypertensive men had greater fat mass, more glucose intolerance, and THE INCIDENCE

F722

0363-6127/90

$1.50

Copyright

0 1990

higher fasting insulin levels than did normotensive controls. When a subgroup of hypertensive men was compared with a subgroup of normotensive controls with similar body mass index values, higher serum insulin levels were still apparent in the hypertensive group. A similar association between hypertension and hyperinsulinemia has also been suggested by others (6, 23). One mechanism by which hyperinsulinemia could predispose toward the development of hypertension is by exerting an antinatriuretic effect on the kidney (9). Atchley et al. (I) observed a marked increase in sodium excretion following abrupt withdrawal of insulin therapy in patients with diabetes mellitus and that reinstitution of insulin therapy decreased sodium excretion. Miller and Bogdonoff (25) also found an antinatriuresis after insulin administration in normal subjects undergoing either solute or water diuresis, and insulin has also been found to decrease sodium excretion in fasted obese subjects (35). The exact mechanisms by which insulin causes antinatriuresis are not completely clear, but have been suggested to be related to a direct effect of insulin on renal sodium reabsorption (9). Insulin infusion reduces sodium excretion in the isolated kidney (26), and intrarenal insulin infusion at rates that do not markedly alter systemic plasma concentrations decreases sodium excretion (8). Micropuncture studies suggest that insulin may increase sodium reabsorption mainly in distal nephron segments or the loop of Henle (8, 22), but recent studies in isolated perfused proximal tubules suggest that insulin may also increase reabsorption in this nephron segment as well (3). Theoretically, a sustained antinatriuretic effect of insulin could lead to chronic hypertension by expansion of extracellular fluid volume. According to this concept, an initial retention of sodium would tend to increase extracellular fluid volume, thereby raising cardiac output and arterial pressure. As arterial pressure increased, the antinatriuretic effect of insulin would be offset by the pressure natriuresis mechanism (19); under steady-state conditions, sodium balance would be only slightly elevated and sodium excretion would be maintained equal to intake, but this would occur at the expense of chronic hypertension. Although the insulin-sodium-blood pressure interrelationship offers an attractive explanation for the link between obesity and hypertension, evidence supporting this concept has been derived mainly from acute studies demonstrating an antinatriuretic effect of insulin or from the American

Physiological

Society

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HYPERINSULINEMIA

AND

BLOOD

studies demonstrating a correlation between insulin and blood pressure in hypertensive patients. A cause and effect relationship between hyperinsulinemia and hypertension has not been demonstrated experimentally. Also, it is not clear whether hyperinsulinemia is capable of causing the sustained reductions in renal excretory capability necessary to cause hypertension or whether other compensatory mechanisms may override its antinatriuretic action before an elevation in blood pressure is required to maintain sodium balance. The main goals of the present study were to determine whether chronic hyperinsulinemia, comparable to that found in obesityassociated hypertension, causes sustained increases in arterial pressure or potentiates the hypertensive actions of angiotensin II (ANG II). All of our studies were conducted in conscious dogs with kidney mass reduced in order to increase their susceptibility to a chronic hypertensive stimulus. Previous studies indicate that the hypertensive effect of other antinatriuretic hormones, such as ANG II or aldosterone, is exacerbated by reducing kidney mass (see Ref. 7 for review). METHODS

Experiments were conducted in conscious mongrel dogs weighing 18.2-31.2 kg (average 23.4 t 0.9 kg). All surgical procedures were conducted under aseptic conditions and pentobarbital sodium anesthesia. The left kidney was exposed through a retroperitoneal flank incision, the poles of the kidney were surgically removed, and the remaining ends were cauterized. Clotting was facilitated by topical application of thrombin (1,000 U/ ml) and application of Gelfoam (Upjohn, Kalamazoo, MI). During surgical removal of the poles of the kidney and cauterization, the renal artery was occluded to aid in hemostasis; the total time of renal artery occlusion was usually 1-2 min. After a 4- to 5wk recovery period, the entire right kidney was removed to produce a reduction in total kidney mass to -30% of normal. Catheters (Tygon, Norton Plastics and Synthetics Division, Akron, OH) were implanted in the femoral arteries and veins for measurement of arterial pressure and intravenous infusions. The catheters were tunneled subcutaneously, exteriorized in the scapular region for protection, and filled with heparin solution (1,000 USP units/ml). The dogs were permitted to recover from surgery, antibiotics were administered daily, and rectal temperatures were measured to ensure that the dogs were afebrile before studies were begun. After l-2 wk of recovery from the second surgical procedure, the dogs were placed in individual metabolic cages in a quiet air conditioned room with a 12-h light cycle and fitted with harnesses containing pressure transducers (Statham Medical Instruments, Hato Rey, PR) mounted at heart level. One of the femoral artery catheters was connected to the pressure transducer so that mean arterial pressure could be continuously recorded on a grass polygraph (model 7D). Mean arterial pressure signals from the Grass recorder were sent to an analog-to-digital converter and analyzed with a digital computer (Turbo XT, PCs Limited, Austin, TX). Analog signals for the blood pressures monitored on the poly-

PRESSURE

REGULATION

F723

graph were sampled 50 times each minute and digitized to provide an average value for each minute throughout the day. The average pressure for each day was then calculated from pressures recorded over an 18-h period between 1400 and 0800 h. To infuse the various solutions continuously, one of the femoral venous catheters was connected to a roller infusion pump (model 375A, Sage Instruments, Cambridge, MA). All solutions were pumped through a disposable Millipore filter (Cathivex, Millipore, Bedford, MA) 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 cage. The dogs were fed two cans (447 g/can) of a sodium-deficient diet (H/D Hills Pet Products, Topeka, KS) that provided -7 meq sodium and 64 meq potassium/day as well as 5 ml of a vitamin syrup (VAL Syrup, Fort Dodge Laboratories, Fort Dodge, IA). Total sodium intake was maintained constant throughout the study by intravenous infusion of sterile isotonic saline at various rates. Before control measurements were started, the dogs were trained to lie quietly while blood samples were obtained from the arterial catheters and studies of renal function were performed beginning at approximately 8:OO each day, 18-20 h after the last feeding. Experimental

Protocol

Insulin infusion in dogs maintained on moderately high sodium intake. In four dogs, total sodium intake was maintained at -138 meq/day by infusion of sterile isotonic saline. In addition, 50 ml/day of sterile water was infused with a syringe pump (Harvard Apparatus, Millis, MA), and -420-830 ml/day 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 7-10 days of control measurements, an intravenous infusion of insulin was started at a rate of 0.5-1.0 mU kg-l. min-’ 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 (see below). The total volume of glucose solution infused was the same as the volume of vehicle infused during the control period. During the first day of insulin infusion measurements of blood glucose were made frequently with a blood glucose monitor (ACCU-CHEK II, Blood Glucose Monitor, Boehringer Mannheim, Indianapolis, IN) 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 after feedings; 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 4 more days. Insulin infusion during very high sodium intake. Seven

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F724

HYPERINSULINEMIA

AND

BLOOD

dogs were maintained on a very high sodium intake, averaging 319 meq/day, by continuous intravenous infusion of isotonic saline. In addition, the dogs also received intravenous infusions of 50 and 710-1,040 ml/day of sterile water as the vehicles for insulin and glucose infusions. After 7-10 days of control measurements, insulin was infused at a rate of 1.0 mu. kg-‘. min-’ for 28 days while plasma glucose was held constant by intravenous infusion by 50% glucose. The total volume infused remained the same as during the control period. After 28 days of insulin infusion, postcontrol measurements were made for 7 days while the dogs were infused with sterile water instead of insulin and glucose. Insulin infusion during ANG II hypertension. In five dogs maintained on an average sodium intake of 325 meq/day, ANG II was infused at a rate of 2.0 ng kg-‘. min-’ for 7 days while control measurements were made. Then an intravenous infusion of insulin was started at 1 .O mu. kg-’ min-l and continued for 28 days while plasma glucose was held constant by intravenous infusion of 50% glucose solution. The total volume infused remained the same as during the control period in which the sterile water vehicles were infused. ANG II infusion was continued throughout the experiment at a rate of 2 ng kg-’ . min. Post-control measurements were made for an additional 5-7 days after stopping insulin and glucose infusions while continuing the ANG II infusion. In three of the dogs, the insulin and glucose infusions were stopped at 7, 17, or 19 days of infusion due to technical difficulties. Glucose clamping. The euglycemic glucose infusion rate per kilogram body weight required during insulin infusion was calculated with the use of a mathematical model of insulin-glucose dynamics. Quantitative relationships (13, 17, 32), scaled to the canine, included stimulation of total tissue glucose uptake by insulin, insulin clearance, and the apparent volumes of distribution of insulin and glucose. Glucose infusions of 7 and 14 rng- kg-‘.rnin-’ were scheduled for insulin infusion rates of 0.5 and 1.0 mU . kg-’ min. l

l

l

l

Analytical

Methods

Glomerular filtration rate and effective renal plasma flow were determined from total clearances of [ ““I]iothalamate (Glofil, Isotex Diagnostics, Friendwood, TX) and [1311]iodohippurate (Hippuran, Mallinckrodt Nuclear, St. Louis, MO), respectively, as previously described (20). Plasma and urine sodium and potassium concentrations were determined by flame photometry (IL443, Instrumentation Laboratories, Lexington, MA). Plasma and urine chloride concentrations were measured by coulometric titration (Haake-Buchler Chloridometer, Saddlebrook, NJ). Plasma protein concentration was measured by refractometry (American Optical, Buffalo, NY), and plasma glucose concentration was measured by the hexokinase method (Sigma Diagnostics, St. Louis, MO). Plasma renin activity was measured by the radioimmunoassay method of Haber et al. (18) using lz51labeled angiotensin I from New England Nuclear (Boston, MA) and antibody from Chemicon (El Segundo, CA). Aldosterone was extracted from plasma with 7 vol

PRESSURE

REGULATION

of dichloromethane, and the dried extract was reconstituted with phosphate-gelatin buffer and measured by radioimmunoassay using ?-labeled aldosterone from Amersham (Arlington Heights, IL) and liquid phase antibody from Diagnostic Products (Los Angeles, CA). Plasma insulin was measured by radioimmunoassay using a kit from Cambridge Medical Diagnostics (Billerica, MA). Statistical

Analyses

Experimental data were compared with control data by analysis of variance and, when appropriate, with Dunnett’s t test for multiple comparisons (10). Statistical significance was considered to be P < 0.05. All data are expressed as means t SE, unless otherwise indicated. RESULTS

Insulin Infusion During High Sodium Intake

Moderately

Table 1 shows the effects of 7 days of insulin infusion in dogs with reduced kidney mass and maintained on a sodium intake of 138 meq/day. Mean arterial pressure decreased from 91 & 3 to 84-85 mmHg during the first 2 days of insulin infusion. During the next 5 days of insulin infusion, mean arterial pressure was not significantly different than control. Urine sodium excretion decreased from a control value of 134 t 14 meq/day to 102 $- 8 and 92 +- 16 meq/day, respectively, during the first 2 days of insulin infusion and then returned toward control; during the entire 7 days of insulin infusion, sodium excretion averaged 118 t 7 meq/day. Thus there was a net retention of approximately 112 meq of sodium during 7 days of insulin infusion. Insulin infusion also caused reductions in urine volume and potassium and chloride excretion. The net water retention averaged -760 ml during 7 days of insulin infusion. Plasma insulin concentration increased from a control value of 8.6 t 0.7 to 42-53 &J/ml during 7 days of insulin infusion (Table 1). Plasma glucose concentration remained relatively constant, averaging 128 t 6 mg/lOO ml during control and 114 t 16 mg/lOO ml during insulin infusion for 7 days. Plasma renin activity and aldosterone concentration were suppressed to low levels during the control period because of the liberal sodium intake and were not significantly changed during insulin infusion. Insulin

Infusion

During

Very High Sodium Intake

Insulin infusion for 28 days did not increase mean arterial pressure in dogs maintained on very high sodium intake (Fig. 1). In fact, there were small but consistent reductions in mean arterial pressure during the first several days of insulin infusion. Thereafter, mean arterial pressure gradually returned toward control and after 28 days was not significantly different from control. However, on the 1st day after stopping insulin infusion there was a small but consistent increase in mean arterial pressure. Urinary sodium excretion decreased from a control

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HYPERINSULINEMIA TABLE

- _1. Effects

of insulin MAP,

Control (mean, 2-4 days) Insulin Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Post-control (mean, 4 days)

infusion

AND

in dogs with

mmHg

UN& medday

UC& meday

91t3

134t14

85t2 84tl 9224 92t3 90t2 87k2 87tl 88&l

102t8 92tl6 130t5 159t14 128kll 105t5 109t22 13O_t6

BLOOD

PRESSURE

reduced kidney

U&, n-w/day

ml/day

181t14

58k2

1,830&127

133t9 129*23 173t9 2Olzk24 180t19 160tlO 172220 168t10

36t5 4Ok8 40*7 38t9 46tll 54210 70t2 65zk8

1,263+72 1,265+139 1,580&60 1,604+137 1,444+114 1,459+64 1,525*188 1,737+8

v,

F725

REGULATION

mass P glucose7 mg/lOO

Pinsulin,

U/ml

ml

P meK&/l

P protein, g/100 ml

PRA, ng ANG I. ml-‘. h-l

ALDO, ng/lOO ml

8.6t0.7

128t6

5.12t0.04

6.71t0.26

0.14t0.04

1.7tO.l

53.9t12.9

134t16

4.49t0.18

6.43t0.26

0.28t0.08

1.520.2

43.2t10.4

lOOt18

5.06t0.10

6.20k0.24

0.2OkO.05

1.9t0.3

42.2t9.3 13.7k3.0

109k16 122k6

5.1420.12 4.82kO.10

6.09t0.13 6.66t0.13

0.24t0.04 0.23zO.09

4.9t2.5 2.5t0.5

Values are means & SE; n = 4 dogs. Insulin MAP, mean arterial pressure; was infused at 0.5-1.0 mU kg-‘. min? excretion; UclV, urinary chloride excretion; UkV, urinary potassium excretion; V, urine volume; Pinsulin, plasma insulin plasma glucose concentration; Pk, plasma potassium concentration; Pprotein, plasma total protein concentration; PRA, ALDO, plasma aldosterone concentration. HIGH

SODIUM

+ $

KIDNEY

UNa\j, urinary concentration; plasma renin

sodium Pglucose, activity;

MASS

120

MEAN ARTERIAL PRESSURE

FIG. 1. Effects of insulin infusion for 28 days on mean arterial pressure in dogs with reduced kidney mass maintained on an average sodium intake of 319 meq/ day. n = 7 dogs.

100

mmw

90

80

0

4

8

12

TIME

16

20

24

28

32

(days)

value of 304 t 10 to 261 t 18 meq/day during the 1st day of insulin infusion (Fig. 2). Although there was some variability in sodium excretion, cumulative sodium balance increased by 305 t 53 meq after 14 days of insulin infusion; thereafter, cumulative sodium balance declined, averaging 232 t 22 meq after 28 days of insulin infusion. Potassium excretion decreased markedly during the first 7 days of insulin infusion and then returned toward control. For the entire 28 days of insulin infusion, potassium excretion averaged 62.4 t 1.7 meq/day, compared with a control value of 73.5 t 1.5 meq/day; since potassium intake remained constant, there was a net retention of -310 meq of potassium during the 28 days of insulin infusion. Urine chloride excretion and urine volume paralleled the changes in urinary sodium excretion. Insulin infusion increased glomerular filtration rate significantly by 12-27% (Fig. 3). Effective renal plasma flow also tended to increase (7-11%) during the first 10 days of insulin infusion, but these changes were not

.

statistically signnicant. Renal vascular resistance, calculated as arterial pressure/[ (effective renal plasma - hematocrit)], tended to decrease during infloW(l sulin infusion but the changes were not statistically significant. Plasma insulin concentration increased more than sixfold, averaging 9.8 t 1.5 pU/ml during control and 65.1 -+ 5.2 pU/ml during 28 days of insulin infusion (Table 2). Plasma glucose concentration decreased slightly during insulin infusion but remained within the normal range, averaging 99 t 10 mg/lOO ml during 28 days of insulin infusion. Plasma renin activity was undetectable during the control period and remained suppressed during 28 days of insulin infusion. Plasma aldosterone concentration did not change significantly during 28 days of insulin infusion. Plasma potassium concentration did not change significantly during the first 3 days of infusion, but then increased slightly on days 6-28 of insulin infusion; after 28 days of insulin infusion, plasma potassium . P

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F726

HYPERINSULINEMIA HIGH SODIUM

SODIUM EXCRETION (mEq/day)

300-

+ + KIDNEY

AND

BLOOD

PRESSURE

REGULATION

MASS

HIGH

[

GLOMERULAR FILTRATION RATE (% Control)

2oo

SODIUM

+ + KIDNEY

MASS

120

100

80

I

~;~;~TIVE

60-

r-k%MA (% Control)

POTASSIUM EXCRETION (mEq/dw)

120

100

80

I

I

II

(‘ .‘)20I1098

130

I I I I

-1

1

rnlimin

I

T

110 RENAL VASCULAR RESISTANCE (% Control)

URINE VOLUME (ml/day)

3000

2000

II

90

70 -4

0

8

12

16

TIME

(days)

20

24

28

‘111 32

I

3. Effects of insulin infusion on glomerular filtration rate, effective renal plasma flow, and renal vascular resistance in dogs with reduced kidney mass maintained on an average sodium intake of 319 meq/day. C, average control value t SE for each variable; n = 7 dogs. FIG.

-4

d

:2

llti

TIME

(days)

2'0

:4

:8

32

FIG. 2. Effects of insulin infusion on sodium and potassium excretion and urine volume in dogs with reduced kidney mass maintained on an average sodium intake of 319 meq/day; n = 7 dogs.

increased to 4.73 t 0.16, compared with a control of 4.23 t 0.10 meq/l. There were no significant changes in plasma sodium or chloride concentration during insulin infusion. Plasma protein concentration decreased from a control value of 6.3 t 0.1 g/100 ml to an average of 5.7 t 0.2 g/100 ml during 28 days of insulin infusion. Insulin

Infusion

During

ANG II Hypertension

Arterial pressure averaged 132 t 7 mmHg during the control period in dogs with reduced kidney mass that were maintained on high sodium intake and infused with ANG II (Fig. 4). However, subsequent insulin infusion during continuing ANG II infusion failed to increase mean arterial pressure further; during 28 days of insulin infusion, mean arterial pressure averaged 134 t 5 mmHg. Insulin infusion in ANG II hypertensive dogs also caused a slight retention of sodium (Fig. 5) with sodium excretion decreasing from 319 t 17 to 270 t 25 meq/day on the 1st day of infusion. However, sodium excretion returned to control during the next few days and for the entire 28 days of insulin infusion averaged 314 t 22 meq/ day, a value not significantly different than control. Potassium excretion fell markedly, averaging 47-68% of control during the first 6 days of infusion; thereafter, potassium excretion returned toward control. Urine volume and chloride excretion paralleled the changes in sodium excretion.

Glomerular filtration rate increased lo-13% above control on days l-l 0 of insulin infusion, although the increase was statistically significant only on the 6th day of infusion (Fig. 6). Effective renal plasma flow increased and renal vascular resistance decreased slightly, but the changes were not statistically significant. Plasma insulin concentration increased from 9.7 t 0.7 to 67.3 t 4.5 pU/ml during 28 days of insulin infusion (Table 3). Plasma glucose concentration decreased significantly during insulin infusion but remained within the normal range. Plasma renin activity was below the detectable limits of our assay during the control period and remained undetectable throughout the 28 days of insulin infusion. Plasma aldosterone concentration also remained low, averaging 6.3 t 1.5 ng/lOO ml during control and 4.2 t 1.0 ng/lOO ml during 28 days of insulin infusion. Insulin infusion also caused no significant changes in plasma concentrations of potassium, sodium, or chloride, although plasma protein concentration decreased from 7.25 t 0.22 to an average 6.40 -t 0.25 g/100 ml during 28 days of insulin infusion. DISCUSSION

Hyperinsulinemia

and Blood Pressure

Regulation

The results from these studies indicate that hyperinsulinemia, lasting for as long as 7-28 days at levels comparable to those found in obese hypertensives, did not elevate mean arterial pressure in dogs with reduced kidney mass, even when they were maintained on high

Downloaded from www.physiology.org/journal/ajprenal at Macquarie Univ (137.111.162.020) on February 14, 2019.

HYPERINSULINEMIA

AND

BLOOD

PRESSURE

F727

REGULATION

2. Effects of insulin infusion in dogs with reduced kidney mass and maintained on very high sodium intake

TABLE

P mel(qjl

Control Insulin Day 1 Day 3 Day 6 Day 10 Day ,14 Day 21 Day 28 Postcontrol

P Nap mm

P protein, g/100 ml

4.23zkO.10

144.5t1.0

6.34t0.11

4.19t0.15 4.27t0.20 4.7020.23 4.99t0.21 5.07IkO.18 4.78k0.15 4.73t0.16 4.82t0.14

144.8kl.l 145.8t0.8 145.9t0.6 145.4t0.9 145.220.8 145.7kO.7 144.5t0.7 143.120.5

Values are means t SE; control concentration; P Na, plasma sodium concentration; Pinsulin, plasma insulin

is the average Concentration;

concentration; ANG

ng ANG

6.04kO.14 5.61t0.15 5.34rtO.20 5.29t0.23 5.47t0.19 5.81kO.20 5.96t0.20 7.03t0.24 of 2 days; n = 7 dogs.

PRA, I. ml-l

ALDO, ng/lOO ml

- h-l

0.08~0.02

1.6kO.l

0.17t0.12 0.24kO.17 0.19t0.11 0.18t0.09 0.09*0.04 0.04t0.01 0.05t0.02 0.03t0.01

1.3t0.2 1.3t0.2 1.5t0.2 1.750.3 1.5t0.2 1.3tO.l 1.2t0.1 1.520.1 at 1.0 mU

Insulin was infused Pprotein, plasma protein concentration; PRA, plasma P glucose,plasma glucose concentration.

II + HIGH

SODIUM

+ + KIDNEY

renin

P glucose, mg/lOO

Pinsulin,

dJ/ml

9.8k1.5 78.4t8.3 73.0t7.7 56.2t5.2 55.7t4.2 62.1k8.7 61.1k3.0 69.0t5.7 12.1t1.4 kg-’ l rein-1. activity;

ALDO,

ml

131*4 111t12 104t9 99t15 84t13 110t20 go*11 96t14 12328 Pk, plasma potassium plasma aldosterone

MASS

t FIG. 4. Effects of insulin infusion on mean arterial pressure in dogs infused with ANG II (2.0 ng kg-‘. min-I) with reduced kidney mass maintained on an average sodium intake of 325 meq/day. ANG II infusion was started 7 days before beginning insulin infusion and continued throughout experiment. Numbers at bottom of graph denote number of dogs studied.

c -I

MEAN ARTERIAL PRESSURE

l

100

-

(mml-w 60

-

-4

0

4

8

12

TIME

16

20

24

28

32

(days)

sodium intake. Unexpectedly, there was actually a small but consistent reduction in blood pressure during the first few days of insulin infusion in dogs on high sodium intake. Arterial pressure eventually recovered to the initial control level during chronic hyperinsulinemia, but when insulin infusion was stopped there was a transient increase in mean arterial pressure, suggesting that compensatory mechanisms may have been activated to offset a hypotensive action of insulin. The mechanisms by which hyperinsulinemia decreased arterial pressure in these experiments are not clear from the present study, but previous acute experiments (36) suggest that insulin may attenuate norepinephrine and ANG II-mediated vasoconstriction of peripheral blood vessels. Whether these effects are present in vivo and persist during chronic hyperinsulinemia apparently has not been previously examined. However, it is interesting to note that insulin did not decrease arterial pressure in ANG IIhypertensive dogs, suggesting that insulin may not have had a powerful chronic effect to blunt ANG II-mediated vasoconstriction in these experiments. The most important finding of the present study is that chronic hyperinsulinemia did not elevate blood pres-

sure, even after we attempted to predispose the dogs toward potential hypertensive actions of insulin by reducing their kidney mass and maintaining them on high sodium intake. Previous studies have shown that the functional reserve of the kidney plays an important role in determining the severity of hypertension resulting from various disturbances that tend to raise blood pressure. For example, large increases in sodium intake cause little or no change in blood pressure as long as kidney function is not impaired (21). However, the same increases in sodium intake raise blood pressure considerably in animals with reduced renal mass (7). In the present study, control values for mean arterial pressure averaged -15 mmHg higher in dogs with reduced kidney mass maintained on 319 meq sodium/day, compared with a sodium intake of 138 meq/day. This salt sensitivity of arterial pressure is not found in normal dogs (21). Likewise, chronic administration of mineralocorticoids causes relatively small increases in blood pressure in normal animals, but much more severe hypertension when renal mass is reduced or when sodium intake is high (7). Thus, by reducing kidney mass and maintaining the dogs on high sodium intake, we expected to greatly exacerbate

Downloaded from www.physiology.org/journal/ajprenal at Macquarie Univ (137.111.162.020) on February 14, 2019.

F728

HYPERINSULINEMIA ANG

II

+ HIGH

SODIUM

++

KIDNEY

AND

BLOOD

PRESSURE

REGULATION

MASS

ANG

II + HIGH

SODIUM

+ + KIDNEY

MASS

I (‘

4n.xi1.9 mllmin I I

GLOMERULAR ;l;;EATlON

300 SODIUM EXCRETION

(mEq/dW

(%

1

110

Control)

200

I I

170-

(‘-

150-

100.016.9 mllmin I I I I I I I IT

E;z;yTIVE 13080POTASSIUM EXCRETION

60

RTEhMA

(%

Control)

-

-

(mEWJay) 40120

wx-w.1,9 mrnllg; mlimin f

100 1h I I

6000-

5000

RENAL VASCULAR RESISTANCE (% Control)

i I

12

4000URINE VOLUME (ml/day)

TIME

16

20

24

28

32

(days)

3000-

-4

0

4

8 TIME

12

16

20

24

28

32

(days)

FIG. 5. Effects of insulin infusion on sodium and potassium excretion and urine volume in dogs infused with ANG II (2.6 ng. kg-‘. min-‘) with reduced kidney mass maintained on an average sodium intake of 325 meq/day. ANG II infusion was started 7 days before beginning insulin infusion and continued throughout the experiment. Numbers at bottom graph denote number of dogs studied.

any hypertensive actions of insulin. Yet even under these conditions chronic hyperinsulinemia did not elevate arterial pressure. Our experiments were also designed to test the possibility that chronic hyperinsulinemia might increase arterial pressure when circulating ANG II was maintained at a constant but slightly elevated level. With many perturbations that tend to increase arterial pressure via increases in sodium balance, such as increased sodium intake or mineralocorticoid excess, there is normally a suppression of renin release and ANG II formation that tends to increase sodium excretion and minimize the rise in blood pressure (21, 27). Since insulin has been postulated to cause hypertension by reducing renal excretory capability (8), it seemed important to determine whether insulin might elevate arterial pressures if ANG II levels were maintained above normal. Because many obese patients with hypertension have normal plasma renin activity, which could represent an inappropriate renin response to volume expansion, the question of whether insulin causes hypertension when ANG II levels are inappropriately elevated may have clinical significance. However, the results from our studies indicate that

FIG. 6. Effects of insulin infusion on glomerular filtration rate, effective renal plasma flow, and renal vascular resistance in dogs infused with ANG II (2.0 ng. kg-‘. min-‘) with reduced kidney mass maintained on an average sodium intake of 325 meq/day. ANG II infusion was started 7 days before beginning insulin infusion and continued throughout experiment. C, average control values t SE for each variable. Numbers at bottom of graph denote number of dogs studied.

chronic hyperinsulinemia did not elevate arterial pressure even when ANG II was maintained at a constant high level by infusion of 2.0 ng . kg-l min-l of ANG II. Thus our results provide no evidence that insulin potentiates the hypertensive effects of ANG II or that the inability to suppress ANG II unmasks a hypertensive action of elevated insulin levels. Although the results from the present study suggest that chronic hyperinsulinemia does not elevate blood pressure and does not potentiate the hypertensive effects of ANG II in dogs, plasma insulin has been reported to be closely associated with hypertension in obese patients. Hyperinsulinemia and hypertension frequently occur in obese patients, and weight reduction in these individuals often causes parallel reductions in blood pressure and plasma insulin (6, 14, 34). However, while there appears to be an association between obesity, hyperinsulinemia, and hypertension, a cause and effect relationship has not been established. If the results of the present study are indicative of the role of insulin in human hypertension, it seems likely that hyperinsulinemia, although closely correlated with blood pressure, may not play a direct causal role in obesity-associated hypertension. However, it is possible that some factor associated with obesity may interact with insulin to raise arterial pressure. Further studies are needed to test this hypothesis. l

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HYPERINSULINEMIA

AND

BLOOD

PRESSURE

F729

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3. Effects of insulin infusion in dogs with reduced kidney mass that were infused with ANG II and maintained on very high sodium intake TABLE

P me;/1

Control insulin Day 1 Day 3 Day 6 Day 1-O Day 14 Day 21 Day 28 Postcontrol

P me;;1

P

protein,

g/100

ml

ng ANG

PRA, I - ml-l

- h-l

ALDO, ng/lOO ml

3.5720.29

143.6kO.5

7.25t0.22

0.02kO.003

4.8t0.9

3.26k0.23 3.24k0.27

144.4tl.O 145.Otl.6 147.6tl.2 146.5t0.6 146.5H.3 144.7t1.0 144.2tl.l 143.0t0.6

6.94kO.21 6.6lt0.21

0.01t0.002 0.01t0.005 0.01~0.005 0.02t0.005 0.01-c-0.006

2.7t0.3 2.7t0.3 3.9t0.7 3.4t0.3 2.520.2 3.2tl.2 2.4t0.2 4.651.1

3.61t0.28

3.80t0.28 3.5lkO.48 4.18t0.22

3.80t0.02 3.48t0.20

6.13t0.27 5.96t0.33 5.9MO.33 6.15t0.20 6.90t0.50

7.03kO.27

0.01t0.010 0.01t0.005 0.01~0.003

Pinsdin,

d-J/ml

P glucose, ng/lOO

ml

n

9.7kO.7

12Ort8

5

92.1t13.4 73.1t2.0

125tl6

5 5 5 4 4 2 2 3

64.8k3.5 53.0t12.8 58.2t8.9 57.2tl.3

57.5t0.6 15.6t3.7

95t9 85t9 108t32 73k22 115t36 108-r-45 138tl9

Values are means t SE; control is the average of 2 days; n, no. of dogs. Insulin was infused at 1.0 mU kg-I-min-‘, ANG II at 2.0 ng- kg-‘min-l. Pk, plasma potassium concentration; Pprotein, plasma protein concentration; PRA, plasma renin activity; P Na, plasma sodium concentration; ALDO, plasma aldosterone concentration; Pglucose, p lasma glucose concentration; Pinsulin, plasma insulin concentration.

Effects of Hyperinsulinemia on Renal Excretion Previous studies have demonstrated that acute insulin administration reduces urinary sodium excretion (8). Using the glucose clamp technique in combination with recollection micropuncture methods, DeFronzo (8) demonstrated that hyperinsulinemia reduced renal sodium excretion by X0% with no changes in glomerular filtration rate and an increase in sodium reabsorption at a site beyond the proximal tubule. Kirchner (22) recently reported that euglycemic insulin administration stimulated chloride reabsorption in the loop of Henle of volume-expanded rats, but had no effect on superficial proximal or distal convoluted tubules. Recent studies in isolated rabbit proximal tubules suggest that insulin added to the basolateral side stimulated fluid transport and caused a more negative transepithelial potential difference (3). Thus it is generally accepted that acute insulin administration directly increases renal sodium reabsorption, although the quantitative importance of the different nephron sites of action of insulin in different physiological and pathophysiological conditions is still unclear. However, the effects of sustained hyperinsulinemia on renal excretion have not been widely investigated. The results from the present study in which the chronic renal effects of insulin were examined are consistent with previous reports that acute administration of insulin causes sodium and water retention mainly by increasing sodium reabsorption. We found no reductions in glomerular filtration rate associated with insulin-mediated antinatriuresis; in fact, there were small but consistent increases in glomerular filtration rate during the periods of insulin-mediated sodium retention.-After several days of hyperinsulinemia there was an escape from sodium and water retention and a return of urine excretion toward normal. The mechanisms responsible for the renal escape during chronic hyperinsulinemia are unclear, but do not appear to be secondary to pressure natriuresis since arterial pressure did not increase with insulin infusion. In fact, there was actually a small decrease in blood pressure that may have contributed, in part, to the mild sodium retention observed during insulin infusion in dogs maintained on a high sodium intake. Suppression of ANG II and aldosterone forma-

tion also cannot explain the escape from sodium retention since plasma renin activity and plasma aldosterone concentration did not change during insulin infusion, possibly because they were already suppressed to low levels due to a high sodium intake. Also, in dogs infused with ANG II in order to maintain circulating levels constant, the escape from sodium retention paralleled that observed in other experiments. One potential mechanism that could be important in offsetting the effects of insulin on tubular reabsorption is the increase in glomerular filtration rate that occurred with hyperinsulinemia; glomerular filtration rate was elevated by lo27% during insulin infusion even though effective renal plasma flow was not markedly increased. The exact mechanisms by which insulin raises glomerular filtration rate and the role of other factors besides increased glomerular filtration rate in offsetting the antinatriuretic effects of insulin are still uncertain. The decreases in potassium excretion observed during chronic insulin infusion were even more dramatic than the reductions in sodium excretion. Potassium excretion decreased by >50% and the reduction was maintained for 6-7 days in all groups of dogs. Although the mechanisms responsible for the antikaliuresis were not the major focus of the present study, previous acute experiments suggest that insulin-mediated reductions in potassium excretion may be secondary to hypokalemia caused by potassium uptake in extrarenal tissues (9, 30). However, in the present study we were unable to demonstrate consistent reductions in plasma potassium concentration during chronic hyperinsulinemia. It should be noted, however, that our first measurements of plasma potassium concentration were made -18 h after beginning insulin infusion and that hyperinsulinemia could have caused a transient reduction in plasma potassium concentration lasting for only the first few hours. However, it seemsunlikely that the sustained reductions of potassium excretion, lasting for as long as 7 days, can be explained by decreased plasma potassium concentration. A reduction in potassium excretion also cannot be explained by decreased aldosterone secretion, since plasma aldosterone concentration did not change significantly during chronic insulin infusion. A third possibility is that hyperinsulinemia may increase proximal tubular or loop

Downloaded from www.physiology.org/journal/ajprenal at Macquarie Univ (137.111.162.020) on February 14, 2019.

F730

HYPERINSULINEMIA

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of Henle sodium chloride reabsorption, thereby decreasing volume flow to the distal nephron which, in turn, would tend to reduce potassium excretion. A reduction in distal nephron flow rate would also tend to activate a macula densa feedback mechanism that would tend to increase glomerular filtration rate in an attempt to return distal delivery toward normal (3 1)) possibly explaining the rise in glomerular filtration rate observed during insulin infusion in our studies. Although previous studies have suggested that insulin may raise volume reabsorption in isolated perfused proximal tubules (3) and chloride reabsorption in the loop of Henle (22), there have been no studies, to our knowledge, that have examined the effects of chronic hyperinsulinemia on distal sodium chloride delivery. Further studies are needed to elucidate the mechanisms responsible for the long-term effects of insulin on control of renal hemodynamics and electrolyte excretion. Insulin

Resistance

and Hyperinsulinemia

The cause of hyperinsulinemia in obesity-associated hypertension is uncertain. One widely accepted concept is that hyperinsulinemia is a compensation secondary to a blunted response of peripheral receptors to the effects of insulin on glucose transport (i.e., insulin resistance). However, the precise mechanisms responsible for insulin resistance have not been established. One possibility is that obese individuals have periodic bursts of insulin release due to overeating and that frequent hyperinsulinemia eventually leads to a “downregulation” of insulin receptors linked to glucose transport. Over a long period of time, this could result in the need for higher levels of insulin to maintain normal glucose homeostasis, even during periods of fasting. When various cells, including hepatocytes, lymphocytes, and adipocytes are cultured in media containing insulin, they exhibit a time-dependent decrease in the concentration of insulin receptors (5, 16). Also, the number of insulin receptors on cells acutely removed from patients with a variety of diseases appears to correlate inversely with the insulin concentrations to which these cells have been exposed in vivo (2). However, a decrease in the number of insulin receptors does not necessarily imply a less effective biological response to insulin because there appears to be a substantial number of “spare receptors” for insulin. That is, a far greater number of insulin receptors are normally present than are needed to generate a maximal insulin response (15). In support of the possibility that high levels of insulin may cause significant insulin resistance, Maragou et al. (24) reported that infusion of insulin for 20 h in normal humans, while maintaining plasma glucose constant, caused a considerable decrease in insulin sensitivity, judging from glucose tolerance tests administered after insulin infusion was stopped. Although the present study was not designed specifically to analyze the mechanisms of insulin resistance, our observations do provide some insight concerning the question of whether insulin resistance occurs secondarily to high levels of plasma insulin. Our results provided no indication that the rate of glucose infusion needed to maintain euglycemia decreased during hyperinsulinemia,

PRESSURE

REGULATION

lasting for 7-28 days. In fact, plasma glucose concentration tended to decrease slightly during chronic hyperinsulinemia with a constant rate of glucose infusion, suggesting that progressively higher rates of glucose infusion may be necessary to maintain euglycemia. Since the rate of glucose infusion equals net glucose uptake by all tissues of the body under steady-state conditions of euglycemia, the rate of glucose infusion needed to maintain normal plasma glucose provides a measure of whole body tissue sensitivity to exogenous insulin. Presumably, if peripheral sensitivity to insulin decreases gradually with chronic hyperinsulinemia, then this should be manifest by a gradual decrease in the rate of glucose infusion needed to maintain euglycemia. However, even after 28 days of hyperinsulinemia, the rate of glucose infusion needed to maintain euglycemia was just as high as during the first 24 h of hyperinsulinemia. Although it is possible that downregulation of insulin receptors may have occurred if hyperinsulinemia had been sustained for more than 28 days, it seems likely that the insulin resistance observed in obesity is more complicated than a simple downregulation of insulin receptors in response to sustained increases in plasma insulin concentration. Another possibility is that insulin resistance occurs as a primary disturbance in obese hypertensives and that hyperinsulinemia is a compensatory mechanism aimed at overcoming insulin resistance. Recently, Ferranini et al. (12) have reported that insulin resistance occurs in patients with essential hypertension independent of obesity and glucose intolerance. However, further studies are needed to determine the precise causes of insulin resistance and whether there is a causal relationship between insulin resistance and hypertension. In summary, we found no evidence that chronic hyperinsulinemia, comparable to that found in obesityassociated hypertension, causes sustained increases in arterial pressure or potentiates the hypertensive effects of ANG II. Insulin infusion did, however, cause significant sodium and water retention and even more dramatic reductions in urinary potassium excretion. In addition, hyperinsulinemia also caused increases in glomerular filtration rate that may be important in offsetting the effects of insulin on sodium reabsorption. These observations suggest that chronic hyperinsulinemia per se cannot account for obesity-associated hypertension and that additional factors may be much more important. We thank Gwendolyn Robbins and Ivadelle Heidke for excellent secretarial assistance and William Dixon and Cheri Coody for technical assistance. This work was supported by National Heart, Lung, and Blood Institute Grants HL-39399, HL-11678, and HL-23502. Part of this work has been published in preliminary form (Am. J. Hypertens. 2: 171-173, 1989). Address for reprint requests: J. E. Hall, Dept. of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS 39216-4505. Received

17 March

1989; accepted

in final

form

21 September

1989.

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