In vivo insulin
action in genetic models of hypertension
SIMONA FRONTONI, LYNNE OHMAN, JOSEPH R. HAYWOOD, RALPH A. DEFRONZO, AND LUCIANO ROSSETTI Departments of Medicine and Pharmacology, University of Texas Health Science Center and Audie Murphy Veterans Hospital, San Antonio, Texas 78284 Frontoni, Simona, Lynne Ohman, Joseph R. Haywood, Ralph A. DeFronzo, and Luciano Rossetti. In vivo insulin action in genetic models of hypertension. Am. J. Physiol. 262 (Endocrinol. 1Metab. 25): E191-E196,1992.-Insulin resistance has been described in nonobese subjects with essential hypertension. At present it is unknown whether hypertension per se may lead to the onset of insulin resistance. To examine this question we studied in vivo insulin action in two rat models of genetic hypertension. Four groups of conscious rats were studied: Milan hypertensive (MHS), Milan normotensive (MNS), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY). Mean arterial pressure was increased in SHR vs. WKY in both the fed (184 t 5 vs. 126 t 6 mmHg; P < 0.001) and fasting (160 t 5 vs. 129 t 5; P < 0.001) states. During highdose insulin clamps, total body glucose uptake (mg kg-l. min-‘) was similar in MNS (28.7 t 1.4) vs. MHS (33.6 t 3.0) and in WKY (34.6 t 1.8) vs. SHR (35.7 t 2.4). During low-dose insulin clamps, suppression of hepatic glucose production (3.5 t 0.6 vs. 3.0 & 0.5 mg* kg-l. min-l) and stimulation of glycolysis (12.9 t 0.8 vs. 14.4 t 1.5 mg=kg-’ l rein-‘) were similar in WKY vs. SHR, whereas glucose uptake (24.6 t 1.9 vs. 18.3 t 1.2 mg= kg-l. min-‘; P < 0.01) and muscle glycogenic rate (10.2 t 1.1 vs. 6.5 rfr 1.1 mg* kg-lomin-l; P < 0.05) were increased in SHR vs. WKY. In conclusion, 1) feeding markedly augments blood pressure in hypertensive but not in normotensive rats, and 2) hepatic and muscle insulin sensitivity are normal or increased in two different rat models of genetic hypertension. These results provide evidence that high blood pressure per se does not invariably lead to the development of insulin resistance. l
glycogen synthesis;
insulin
clamp; insulin
sensitivity;
glycolysis
AND INSULIN RESISTANCE are common findings in subjects with essential hypertension (2, 7, 15, 16, 18, 24, 35, 37, 38, 41). The observation that impaired sensitivity to insulin is present in lean hypertensive individuals with normal glucose tolerance (2, 7, 24, 35) suggests one of three possibilities as follows: 1) hypertension represents the primary disturbance and secondarily leads to the development of insulin resistance, 2) insulin resistance represents the primary disorder and secondarily leads to the development of hypertension, and 3) the two disorders are coinherited and develop in a parallel, yet unrelated, fashion. The treatment of hypertension may either lead to a worsening (20, 25, 33-35, 37, 46) or an improvement (14, 25, 26) in glucose tolerance and insulin sensitivity, depending on the therapeutic regimen that is selected. Therefore it is difficult to dissect out the impact of high blood pressure per se on insulin-mediated glucose metabolism from these previous human studies. Animal models have been used extensively to characterize several aspects of the pathogenesis of insulin resistance (3, 4, 8, 11, 12, 21, 22, 2%31,36). A similar approach has been proposed for the association of high blood pressure and insulin resistance HYPERINSULINEMIA
0193-1849/92
$2.00 Copyright
(9, 19, 40, 42). According to this line of reasoning, if the decreased sensitivity to insulin described in hypertensive individuals is the consequence of a metabolic or hemodynamic alteration due to elevated blood pressure, one would expect to observe an impairment in insulin sensitivity in animal models of genetic hypertension. Glucose tolerance has been evaluated in spontaneously hypertensive rats (SHR) and compared with their normotensive strain-matched control, the Wistar-Kyoto rat (WKY) (9, 19, 40, 42, 43). Although glucose tolerance was impaired in SHR compared with WKY in the majority of the previous reports (19, 42, 43), it was unchanged in conscious and unstressed rats (40). Direct measurement of insulin sensitivity in genetic models of hypertension has provided contradictory results. Evidence for abnormalities in insulin-mediated glucose metabolism was suggested by Mondon and Reaven (19) and by Hulman et al. (lo), while Tsutsu et al. (40) showed increased insulin sensitivity in SHR. In the present study we examined the action of insulin on whole body glucose metabolism and on the intracellular metabolic pathways of glucose disposal in two genetic models of hypertension. MATERIALS
AND
METHODS
Animals. Two groups of male 24-h fasted rats were studied as follows: group I, Milan normotensive rats (MNS; n = 5), aged 90 & 2 days, and Milan hypertensive rats (MHS; n = 6), aged 94 t 3 days (Farmitalia, Milan, Italy); and group II, Wistar-Kyoto Rats (WKY; n = ll), aged 101 * 3 days, and spontaneously hypertensive rats (SHR; n = 12), aged 101 t 2 days (Charles River Laboratories, Wilmington, MA and/or Harlan, Indianapolis, IN). Mean body weights were similar in MNS compared with MHS (313 t 15 vs. 313 t 7 g) and in WKY compared with SHR (236 t 8 vs. 249 t 4 g). The rats were given free access to food and water and were housed in individual cages in an air-controlled room, which was subjected to a standard 12:12-h light-dark cycle (lights on at 6:00 A.M.). Five to seven days before the experiment, all animals were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body wt), and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery. The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. Both catheters were exteriorized through the skin at the back of the neck. Blood pressure measurement. Five to seven days after catheter placement, blood pressure was evaluated in conscious unrestrained rats under both fasting (24 h) and fed conditions. At 8:00 A.M. the arterial catheter was attached to a Cobe pressure transducer through an extension of polyethylene (PE50) tubing for continuous recording of mean arterial pressure. Heart rate was obtained from the arterial pressure pulse with
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
El91
El92
INCREASED
SENSITIVITY
TO
INSULIN
a cardiotachometer on a Beckman dynograph recorder. After a resting period of at least 30 min, mean arterial pressure and heart rate were obtained in each animal over a 60-min period. Euglycemic clamp study. Insulin-mediated whole body glucose uptake was measured in awake unstressed chronically catheterized rats using the euglycemic clamp in combination with [ 3-3H] glucose infusion as previously described (28-31,39). On the same day of fasting (24 h) blood pressure measurement, rats received an infusion of regular porcine insulin (Eli Lilly, Indianapolis, IN) at 18.0 mU kg-l l rein-’ (MNS, n = 5; MHS, n = 6; WKY, n = 4; SHR, n = 5) or at 3.0 mU kg-l min-’ (WKY, n = 7; SHR, n = 7) for 2 h. A variable infusion of 25% glucose solution was started at time 0 and adjusted to maintain the plasma glucose concentration at -100 mg/dl. A prime (6 &i) continuous (0.4 &i/min) infusion of [3-“HIglucose (New England Nuclear, Boston, MA) was initiated at time -80 min and continued throughout the study. Plasma samples for determination of [3-“HIglucose specific activity were obtained at -80, -50, -35, and -20 min and then at 5- to lo-min intervals throughout the insulin clamp study. Plasma samples for determination of plasma insulin concentration were obtained at time 0,60, and 120 min and for plasma norepinephrine and epinephrine at time 0 and 120 min during the study. The total amount of blood withdrawn during each study was -3.5 ml. To prevent intravascular volume depletion and anemia, an equivalent amount of normal saline (1.5 ml) plus fresh whole blood (2.0 ml blood) obtained by heart puncture from littermates of the test animal was infused at a constant rate (24 pl/min) throughout the study. At the end of the study, rats were injected with pentobarbital (60 mg/kg body wt), the abdomen was quickly opened, and the rectus abdominal and hindlimb muscles were freeze-clamped with aluminum tongs precooled in liquid nitrogen. All tissue samples were kept frozen at -70°C for subsequent analysis. Plasma catecholamines. Mixed venous blood for determination of norepinephrine and epinephrine (1 ml) was drawn into a chilled syringe containing ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA; 25 ~1) and was immediately centrifuged. The plasma was separated from red blood cells and stored at -70°C until assayed. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio, TX. Assays. Plasma glucose was measured by the glucose oxidase method (glucose analyzer; Beckman Instruments, Palo Alto, CA) and plasma insulin by radioimmunoassay using rat and porcine insulin standards. Plasma catecholamine concentrations were measured by the single-isotope radioenzymatic assay described by Peuler and Johnson (23). The sensitivity of this assay for norepinephrine and epinephrine is 20 pg/ml, and the intra- and interassay coefficient of variation (CV) values are 6 and lo%, respectively. Plasma [ 3-3H] glucose radioactivity was measured in duplicate on the supernatants of barium hydroxide-zinc sulphate precipitates (Somogy procedure) of plasma samples after evaporation to dryness to eliminate tritiated water. Muscle glycogen concentration was determined after digestion with amyloglucosidase as previously described (28-30). The intra- and interassay CV values were
action in genetic models of hypertension
SIMONA FRONTONI, LYNNE OHMAN, JOSEPH R. HAYWOOD, RALPH A. DEFRONZO, AND LUCIANO ROSSETTI Departments of Medicine and Pharmacology, University of Texas Health Science Center and Audie Murphy Veterans Hospital, San Antonio, Texas 78284 Frontoni, Simona, Lynne Ohman, Joseph R. Haywood, Ralph A. DeFronzo, and Luciano Rossetti. In vivo insulin action in genetic models of hypertension. Am. J. Physiol. 262 (Endocrinol. 1Metab. 25): E191-E196,1992.-Insulin resistance has been described in nonobese subjects with essential hypertension. At present it is unknown whether hypertension per se may lead to the onset of insulin resistance. To examine this question we studied in vivo insulin action in two rat models of genetic hypertension. Four groups of conscious rats were studied: Milan hypertensive (MHS), Milan normotensive (MNS), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY). Mean arterial pressure was increased in SHR vs. WKY in both the fed (184 t 5 vs. 126 t 6 mmHg; P < 0.001) and fasting (160 t 5 vs. 129 t 5; P < 0.001) states. During highdose insulin clamps, total body glucose uptake (mg kg-l. min-‘) was similar in MNS (28.7 t 1.4) vs. MHS (33.6 t 3.0) and in WKY (34.6 t 1.8) vs. SHR (35.7 t 2.4). During low-dose insulin clamps, suppression of hepatic glucose production (3.5 t 0.6 vs. 3.0 & 0.5 mg* kg-l. min-l) and stimulation of glycolysis (12.9 t 0.8 vs. 14.4 t 1.5 mg=kg-’ l rein-‘) were similar in WKY vs. SHR, whereas glucose uptake (24.6 t 1.9 vs. 18.3 t 1.2 mg= kg-l. min-‘; P < 0.01) and muscle glycogenic rate (10.2 t 1.1 vs. 6.5 rfr 1.1 mg* kg-lomin-l; P < 0.05) were increased in SHR vs. WKY. In conclusion, 1) feeding markedly augments blood pressure in hypertensive but not in normotensive rats, and 2) hepatic and muscle insulin sensitivity are normal or increased in two different rat models of genetic hypertension. These results provide evidence that high blood pressure per se does not invariably lead to the development of insulin resistance. l
glycogen synthesis;
insulin
clamp; insulin
sensitivity;
glycolysis
AND INSULIN RESISTANCE are common findings in subjects with essential hypertension (2, 7, 15, 16, 18, 24, 35, 37, 38, 41). The observation that impaired sensitivity to insulin is present in lean hypertensive individuals with normal glucose tolerance (2, 7, 24, 35) suggests one of three possibilities as follows: 1) hypertension represents the primary disturbance and secondarily leads to the development of insulin resistance, 2) insulin resistance represents the primary disorder and secondarily leads to the development of hypertension, and 3) the two disorders are coinherited and develop in a parallel, yet unrelated, fashion. The treatment of hypertension may either lead to a worsening (20, 25, 33-35, 37, 46) or an improvement (14, 25, 26) in glucose tolerance and insulin sensitivity, depending on the therapeutic regimen that is selected. Therefore it is difficult to dissect out the impact of high blood pressure per se on insulin-mediated glucose metabolism from these previous human studies. Animal models have been used extensively to characterize several aspects of the pathogenesis of insulin resistance (3, 4, 8, 11, 12, 21, 22, 2%31,36). A similar approach has been proposed for the association of high blood pressure and insulin resistance HYPERINSULINEMIA
0193-1849/92
$2.00 Copyright
(9, 19, 40, 42). According to this line of reasoning, if the decreased sensitivity to insulin described in hypertensive individuals is the consequence of a metabolic or hemodynamic alteration due to elevated blood pressure, one would expect to observe an impairment in insulin sensitivity in animal models of genetic hypertension. Glucose tolerance has been evaluated in spontaneously hypertensive rats (SHR) and compared with their normotensive strain-matched control, the Wistar-Kyoto rat (WKY) (9, 19, 40, 42, 43). Although glucose tolerance was impaired in SHR compared with WKY in the majority of the previous reports (19, 42, 43), it was unchanged in conscious and unstressed rats (40). Direct measurement of insulin sensitivity in genetic models of hypertension has provided contradictory results. Evidence for abnormalities in insulin-mediated glucose metabolism was suggested by Mondon and Reaven (19) and by Hulman et al. (lo), while Tsutsu et al. (40) showed increased insulin sensitivity in SHR. In the present study we examined the action of insulin on whole body glucose metabolism and on the intracellular metabolic pathways of glucose disposal in two genetic models of hypertension. MATERIALS
AND
METHODS
Animals. Two groups of male 24-h fasted rats were studied as follows: group I, Milan normotensive rats (MNS; n = 5), aged 90 & 2 days, and Milan hypertensive rats (MHS; n = 6), aged 94 t 3 days (Farmitalia, Milan, Italy); and group II, Wistar-Kyoto Rats (WKY; n = ll), aged 101 * 3 days, and spontaneously hypertensive rats (SHR; n = 12), aged 101 t 2 days (Charles River Laboratories, Wilmington, MA and/or Harlan, Indianapolis, IN). Mean body weights were similar in MNS compared with MHS (313 t 15 vs. 313 t 7 g) and in WKY compared with SHR (236 t 8 vs. 249 t 4 g). The rats were given free access to food and water and were housed in individual cages in an air-controlled room, which was subjected to a standard 12:12-h light-dark cycle (lights on at 6:00 A.M.). Five to seven days before the experiment, all animals were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body wt), and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery. The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. Both catheters were exteriorized through the skin at the back of the neck. Blood pressure measurement. Five to seven days after catheter placement, blood pressure was evaluated in conscious unrestrained rats under both fasting (24 h) and fed conditions. At 8:00 A.M. the arterial catheter was attached to a Cobe pressure transducer through an extension of polyethylene (PE50) tubing for continuous recording of mean arterial pressure. Heart rate was obtained from the arterial pressure pulse with
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
El91
El92
INCREASED
SENSITIVITY
TO
INSULIN
a cardiotachometer on a Beckman dynograph recorder. After a resting period of at least 30 min, mean arterial pressure and heart rate were obtained in each animal over a 60-min period. Euglycemic clamp study. Insulin-mediated whole body glucose uptake was measured in awake unstressed chronically catheterized rats using the euglycemic clamp in combination with [ 3-3H] glucose infusion as previously described (28-31,39). On the same day of fasting (24 h) blood pressure measurement, rats received an infusion of regular porcine insulin (Eli Lilly, Indianapolis, IN) at 18.0 mU kg-l l rein-’ (MNS, n = 5; MHS, n = 6; WKY, n = 4; SHR, n = 5) or at 3.0 mU kg-l min-’ (WKY, n = 7; SHR, n = 7) for 2 h. A variable infusion of 25% glucose solution was started at time 0 and adjusted to maintain the plasma glucose concentration at -100 mg/dl. A prime (6 &i) continuous (0.4 &i/min) infusion of [3-“HIglucose (New England Nuclear, Boston, MA) was initiated at time -80 min and continued throughout the study. Plasma samples for determination of [3-“HIglucose specific activity were obtained at -80, -50, -35, and -20 min and then at 5- to lo-min intervals throughout the insulin clamp study. Plasma samples for determination of plasma insulin concentration were obtained at time 0,60, and 120 min and for plasma norepinephrine and epinephrine at time 0 and 120 min during the study. The total amount of blood withdrawn during each study was -3.5 ml. To prevent intravascular volume depletion and anemia, an equivalent amount of normal saline (1.5 ml) plus fresh whole blood (2.0 ml blood) obtained by heart puncture from littermates of the test animal was infused at a constant rate (24 pl/min) throughout the study. At the end of the study, rats were injected with pentobarbital (60 mg/kg body wt), the abdomen was quickly opened, and the rectus abdominal and hindlimb muscles were freeze-clamped with aluminum tongs precooled in liquid nitrogen. All tissue samples were kept frozen at -70°C for subsequent analysis. Plasma catecholamines. Mixed venous blood for determination of norepinephrine and epinephrine (1 ml) was drawn into a chilled syringe containing ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA; 25 ~1) and was immediately centrifuged. The plasma was separated from red blood cells and stored at -70°C until assayed. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio, TX. Assays. Plasma glucose was measured by the glucose oxidase method (glucose analyzer; Beckman Instruments, Palo Alto, CA) and plasma insulin by radioimmunoassay using rat and porcine insulin standards. Plasma catecholamine concentrations were measured by the single-isotope radioenzymatic assay described by Peuler and Johnson (23). The sensitivity of this assay for norepinephrine and epinephrine is 20 pg/ml, and the intra- and interassay coefficient of variation (CV) values are 6 and lo%, respectively. Plasma [ 3-3H] glucose radioactivity was measured in duplicate on the supernatants of barium hydroxide-zinc sulphate precipitates (Somogy procedure) of plasma samples after evaporation to dryness to eliminate tritiated water. Muscle glycogen concentration was determined after digestion with amyloglucosidase as previously described (28-30). The intra- and interassay CV values were
