Lipolytic sensitivity and response to fasting in normotensive and hypertensive obese humans

Lipolytie Sensitivity and Response to Fasting in Normotensive and Hypertensive Obese Humans R a y m o n d R. T o w n s e n d a n d S a m u e l Klein

Obese persons with hypertension are at greater risk for diabetes and hyperlipidemia than normotensive obese persons. It has been postulated that increased lipolytic rates contribute to these metabolic diseases. Therefore, we evaluated the glycerol rate of appearance (Ra) in plasma, an index of whole-body lipolytic activity, during basal conditions and during 60 minutes of epinephrine infusion after 12 and 84 hours of fasting in six normotensive (body mass index [BMI], 39.9 -+ 1.8 kg/m 2) and six hypertensive (BMI, 38.7 _+ 1.6 kg/m 2) obese persons. Basal glycerol Ra was lower in hypertensive than in normotensive subjects at both 12 hours (1.58 -+ 0.21 v2.27 _+ 0.26 i~mol/kg/min, respectively; P < .01) and 84 hours (2.04 ± 0.06 v2.50 + 0.13 i~mol/kg/min, respectively; P < .01) of fasting. Peak glycerol Ra during epinephrine infusion after 84 hours of fasting (5.69 _+ 0.72 and 11,40-+ 0.78 i~mol/kg/min for hypertensive and normotensive subjects, respectively) was significantly greater than at 12 hours (3.09 -+ 0.29 and 5.06 -+ 0.69 i~mol/kg/min) in both hypertensive and normotensive subjects. However, peak glycerol Ra was lower in hypertensive than in normotensive subjects after 12 and 84 hours of fasting (P < .01 for 84 hours). We conclude that hypertension in obese persons is associated with a decrease in both basal lipolytic rates and lipolytic sensitivity to epinephrine infusion.

Copyright © 1997 by W.B. Saunders Company YPERTENSIVE OBESE PERSONS are at higher risk for metabolic diseases such as diabetes, hyperlipidemi a, and coronary artery disease than normotensive obese persons.l-3 It is possible that alterations in the regulation of lipolysis contribute to the pathogenesis of these metabolic complications by increasing the delivery of fatty acids to the liver and skeletal muscle? as has been suggested in individuals with upper-body obesity. 5,6 However, the relationship between hypertension and lipid kinetics has not been carefully studied. Theoretical arguments can be made to support either upregulation or downregulation of lipolytic activity in obese patients with hypertension. Hypertensive obese persons could exhibit increased whole-body lipolytic rates because of alterations in the major humoral factors that stimulate (catecholamines) and inhibit (insulin) lipolysis in humans. It has been demonstrated that hypertensive obese people have both increased sympathetic nervous system activity7-9 and decreased insulin sensitivity. 7,~° In contrast, hypertensive obese persons could have lower lipolytic rates than normotensive obese persons because of downregulation of [3-adrenergic receptors caused by chronically elevated sympathetic nervous system activity 11 and because of higher circulating insulin concentrations. 7 An understanding of the interrelationship between obesity, hypertension, and lipolytic activity may also have therapeutic implications. Hypocaloric diets and total energy restriction have been used to decrease body fat in obese patients. 12,13 In

H

normotensive lean subjects, short-term fasting causes a considerable increase in the lipolytic rate and lipolytic sensitivity to catecholamine infusion. 14,15 This adaptive response enhances the use of body fat as fuel. However, both the lipolytic response to fasting and lipolytic sensitivity to [3-adrenergic stimulation are blunted in obese individuals, 5 which could hinder efforts to lose body fat by caloric restriction. The known alterations in sympathetic nervous system activity 7-1° and insulin sensitivity 9 associated with hypertension suggest that the adaptive response to fasting may differ between normotensive and hypertensive obese groups. However, the effect of energy restriction on lipolysis and lipolytic sensitivity has not been studied in hypertensive obese subjects. The purpose of the present study was to evaluate in obese subjects the influence of hypertension on lipid metabolism in the basal state and during different physiological conditions that stimulate lipolytic activity. We hypothesized that the increase in [3-adrenergic stimulation and insulin resistance associated with hypertension would increase both basal lipid kinetics and lipolytic sensitivity to [3-adrenergic stimulation. The basal glycerol rate of appearance (Ra) in plasma, a measure of whole-body lipolytic activity, and glycerol Ra during catecholamine infusion, a measure of lipolytic sensitivity, were determined in vivo in hypertensive and normotensive obese subjects during postabsorptive conditions and after 84 hours of fasting. SUBJECTS AND METHODS

Subjects From the Department of Internal Medicine, University of Pennsylvania Medical Center, Philadelphia, PA; and the Department of Internal Medicine, Washington University School of Medicine, St Louis, MO. Submitted October 9, 1996; accepted April 6, 1997. Supported by National Institutes of Health (NIH) Grant No. DK49989, General Clinical Research Center Grant No. RR-O0073, NIH Training Grant No. DK-07006, and administrative/educational funds from the Dialysis Clinics Incorporated Renal Education and Development fund. Address reprint requests to Raymond R. Townsend, MD, University of Pennsylvania Medical Center, 210 White Bldg, 3400 Spruce St, Philadelphia, PA 19104. Copyright © 1997 by W.B. Saunders Company 0026-0495/97/4609-0019503.00/0 1080

Twelve obese subjects with a body mass index (BMI) greater than 30 kg/m2 and a waist to hip ratio greater than 0.90 participated in this study (Table 1). Six subjects had essential hypertension (diastolic blood pressure > 90 mm Hg in the sitting position), and six were normotensive. Hypertensive and normotensive subjects were matched for age and BMI. All hypertensive and three normotensive subjects were men. Each subject was evaluated with a standard examination including a medical history, physical examination, routine laboratory chemistry and urinalysis, and a 12-lead electrocardiogram before study participation. Subjects with diabetes and those taking prescription medication were excluded. Hypertensive subjects either were untreated or had previous antihypertensive therapy discontinued at least 2 weeks before study participation. All subjects provided informed consent before participating in this study, which was approved by the Institutional Review Board and the Metabolism, Vol 46, No 9 (September), 1997: pp 1080-1084

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LIPOLYSIS IN HYPERTENSIVES

Table 1. Subject Characteristics Normotensive (n = 6)

Hypertensive (n = 6)

Age (yr) Height (cm)

Characteristic

32 +_ 4 170 ± 3

33 _+ 2 173 +_ 2

Weight (kg) BMI (kg/m 2)

111.8 _+ 4.1 39,9 + 1.8

119.7 +_ 5.9 38.7 _+ 1.6

115 + 4

134_+ 4

Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (bpm)

75 _+ 2 61 _+ 3

96 +_ 4 74 _+ 3*

NOTE. Values are the mean -- SE.

radioenzymatic assay or high-performance liquid chromatography. 17,18 Plasma insulin was determined by radioimmunoassay (Incstar, Stillwater, MN). Plasma glycerol was determined enzymatically with an automated analyzer using a glycerol kinase method (Technicon, Tarrytown, NY). Plasma nonesterified fatty acid concentrations were quantified by gas chromatography. 19 Isotopic enrichment of plasma glycerol was determined by gas chromatography-mass spectrometry (GC-MS) using an MSD 5971 system (Hewlett-Packard, Palo Alto, CA) with an HP-1 12 × 0.2-mm fused silica capillary column as described previously.2° Derivatized samples were injected into the GC-MS, where ions were formed by electron-impact ionization. Ions with a m/z of 205.1,206.1,207.1, and 208.1 were selectively monitored to determine the tracer to tracee ratio.

Significantly different from corresponding values for normotensive subjects: * P < .05,

General Clinical Research Center of the University of Texas Medical Branch at Galveston.

Protocol

Calculations Basal glycerol Ra was calculated using the Steele equation. 21 During the basal period (45 to 60 minutes), physiologic and isotopic steady state was present, so that Ra = F/TT, where F is the isotope infusion rate in micromoles per kilogram per minute and TT is the tracer to tracee ratio (isotopic enrichment) of glycerol. During epinephrine infusion, the Steele equation for non-steady-state kinetics was used to calculate glycerol Ra. 22 Carbohydrate, lipid, and protein oxidation rates were calculated from measurement of carbon dioxide production, oxygen consumption, and urinary urea nitrogen excretion.23

Subjects were admitted to the General Clinical Research Center at 5:00 PM and ingested a standard meal at 6:00 PM. They began fasting at 8:00 PM on the day of admission and continued until completion of the second infusion study, 86 hours later. All subjects consumed at least 2 L water daily and were given a multivitamin, 60 Meq potassium chloride, and 140 Meq sodium chloride daily. On both of the 2 study days (after 12 and 84 hours of fasting, respectively), intravenous catheters were inserted between 8:00 and 8:30 AM. One catheter was placed in a dorsal hand vein and heated to obtain arterialized venous samples. 16 The other catheter was placed in the opposite forearm to infuse isotopes and epinephrine. After baseline blood samples were obtained at 9:00 AM, a primed (2.2 ~maol/kg) constant (-0.11 ~tmol/kg/min) infusion of [2H5]glycerol was initiated and continued for 120 minutes using a calibrated syringe pump (C.R. Bard, North Reading, MA). The exact isotope infusion rate for each subject was determined by measnring the concentration of isotope in the infusate. Epinephrine was infused at a rate of 0.015 gg/kg/min between 60 and 120 minutes of isotope infusion. Blood samples were obtained at 45, 50, 55, and 60 minutes of [2Hs]glycerol infusion to determine basal glycerol kinetics, and at 65, 70, 75, 80, 85, 90, 100, t10, and 120 minutes to determine the lipolytic effect of epinephrine. Blood was collected in chilled heparinized tubes and placed immediately on ice. The plasma was promptly separated following refrigerated centrifugation, and was stored at - 2 0 ° C until anaIysis. A small aliquot of each sample collected for glycerol was separated for glucose determination. Blood samples for epinephrine, norepinephrine, and insulin assay were obtained at 45, 60, 80, and 100 minutes of isotope infusion. The 45- and 60-minute values were averaged to establish a baseline value. Blood pressure and heart rate were monitored at 10-minute intervals during epinephrine infusion, for safety reasons. Carbon dioxide production, oxygen consumption, and resting energy expenditure were determined after 12 hours of fasting using a metabolic cart (MMC Horizon System; SensorMedic, Anaheim, CA). Measurements were taken immediately preceding catheter placement and between 45 and 60 minutes after beginning the isotope infusion. Values obtained during these two periods were averaged to determine the basal rate for each subject. Urine was collected for 24 hours during the first day of fasting to determine urea nitrogen excretion.

In the postabsorptive state (12-hour fast), resting energy expenditure was similar in both the hypertensive and normotensive groups (16.5 _+ 0.9 v 17.5 _+ 0.7 kcal/kg/h, respectively). However, the respiratory quotient in hypertensive subjects was higher than in normotensive subjects (0.83 _+ 0.02 v 0.77 _+ 0.02, respectively, P < .05). The percentage o f oxidized energy derived from glucose was higher and the percentage derived from fat was lower in hypertensive compared with normotensive subjects (47% _+ 7% v 23% _+ 8% calories derived from glucose oxidation and 53% _+ 7% v 77% _+ 8% calories derived from fat, respectively, P < .05 for both comparisons).

Analysis of Samples

Plasma Hormone Concentrations

Plasma glucose concentration was measured by the glucose oxidase method using a glucose analyzer (Beckman Instruments, Fullerton, CA). Plasma epinephrine and norepinephrine were determined by a

Plasma h o r m o n e concentrations basally, during fasting, and during epinephrine infusion are s h o w n in Table 2. At 12 hours o f fasting, basal plasma insulin concentrations were signifi-

Statistical Analysis All data are presented as the mean _+ SE. The significance o f differences b e t w e e n groups for the demographic variables in Table 1 and the calorimetry data at 12 hours were evaluated using a t test for independent samples. Within- and betweengroup comparisons for data obtained after both 12 hours and 84 hours fasting were analyzed for statistical significance using a two-factor analysis o f variance (ANOVA) for repeated measures. In this two-factor ANOVA, the two independent factors were group (hypertensive or normotensive) and time (12 hours or 84 hours). Data were analyzed using the software program GB-Stat (Dynamic Microsystems, Inc, Silver Spring, MD). Post hoc comparisons were tested for significance using Fisher's least-squares difference (LSD). A P value less than .05 was considered statistically significant. RESULTS

Indirect Calorimetry

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Table 2. Metabolic Parameters During Basal Conditions and During Peak Epinephrine Infusion at 12 Hours and 84 Hours of Fasting Normotensive Variable

Hypertensive

Basal

Peak

Basal

Peak

12-hour fast Insulin (tJU/mL)

15 ± 2

18 ± 3

19 ± 3*

19 ± 2

Epinephrine (pg/mL)

24 ± 3

356 ± 50

76 ± 11t

486 ± 38

Norepinephrine (pg/mL)

182 -+ 15

225 ± 19

344 ± 58*

408 -- 56

Plasma glucose (mg/dL)

91 +_ 5

100 ± 5

94 ± 6

Fatty acids (IJmol/mL) Glycerol Ra (pmol/kg/min) 84-hour fast

0.50 -+ 0,06 2.27 _+ 0.26

Insulin (IJU/mL) Epinephrine (pg/mL) Norepinephrine (pg/mL) Plasma glucose (mg/dL) Fatty acids (pmol/mL) Glycerol Ra (pmol/kg/min)

4 -+ 1§

0.67 ± 0.07 5.06 ± 0.69

101 ± 5

0.51 ± 0.09 1.58 ± 0.21t

7 -+ 1

0.67 ± 0.09 3.09 ± 0.29

8 ± 1§

9 ± 1

42 -+ 5

294 _+ 38

58 ± 5

341 ± 59

168 -+ 21 63 ± 2§

231 ± 29 67 ± 2

303 -+ 61 69 ± 3§

389 ± 57 76 ± 3

1.18 ± 0.08 11.4 -+ 0.78§

0.92 ± 0.115 2.04 ± O.06t§

1.19 ± 0.15 5.69 ± 0.72t$

0.83 _+ 0.085 2.50 ± 0.13

Significantly different from corresponding values for normotensive subjects: *P<.05, I"P< .01. Significantly different from corresponding values at 12 hours of fasting: SP < .05, §P< .01.

cantly higher in the obese hypertensive group. Insulin levels decreased significantly within each group after continued fasting, and plasma insulin remained higher in hypertensive compared with normotensive subjects at 84 hours of fasting. At 12 hours of fasting, basal plasma epinephrine and norepinephrine concentrations were significantly higher in the hypertensive than in the normotensive group. Basal plasma epinephrine decreased with fasting in the hypertensive subjects, but increased in the normotensive group. During epinephrine infusion after 12 hours and 84 hours of fasting, plasma epinephrine concentrations were numerically but not statistically significantly higher in the hypertensive than in the normotensive subjects.

"-" "~"

8

~

6

=t. ~

4

12h

2

Plasma Substrate Concentrations

Basal plasma glucose and free fatty acid concentrations and the response of glucose and fatty acid to epinephrine infusion are shown in Table 2. Basal plasma glucose and free fatty acid concentrations were similar between the hypertensive and normotensive subjects after both 12 hours and 84 hours of fasting. Plasma glucose concentrations decreased significantly during fasting in hypertensive and normotensive (P < .01 at 84 hours v 12 hours within each group). Basal plasma free fatty acid concentrations increased significantly during fasting in both the hypertensive and normotensive groups (P < .05 at 84 h v 12 hours within each group).

•

=

•

•

•

•

•

0

10

20

30

40

50

60

.=. 15 ~

12

~

9

84 h

6 3

Glycerol Kinetics

Basal glycerol Ra and the changes in glycerol Ra with epinephrine infusion are shown in Table 2. Basal glycerol Ra was lower in hypertensive than in the normotensive subjects at both 12 hours and 84 hours of fasting (P < .01 for both time periods). The lipolytic response to epinephrine infusion (glycerol Ra) after 12 hours and 84 hours of fasting is shown in Fig 1. Peak glycerol Ra during epinephrine infusion after 84 hours of fasting was significantly greater than at 12 hours of fasting within both hypertensive and normotensive groups (P < .05 for both). However, peak glycerol Ra was significantly lower in hypertensive than in normotensive subjects only after 84 hours of fasting. The total increase in glycerol Ra during epinephrine

0

0

10

20

30

40

50

60

M i n u t e s of e p i n e p h r i n e i n f u s i o n Fig 1. (Top) Whole-body lipolysis rates (glycerol Ra) before (basal) and following epinephrine infusion (onset marked by down arrow) after 12-hour fast; (0) mean values (-+SE) for hypertensive subjects, and (O) mean values for normotensive subjects. (Bottom) Wholebody lipolysis rates (glycerol Ra) before (basal) and following epinephrine infusion (onset marked by down arrow) after 84-hour fast; (0) mean values for hypertensive subjects, and (O) mean values for normotensive subjects.

LIPOLYSIS IN HYPERTENSIVES

1083

infusion (area between baseline and glycerol Ra curve) tended to be lower in the hypertensive than in the normotensive group at 12 hours of fasting (69 _+ 35 v 89 -+ 18 ~mol/kg/60 rain, respectively), but the differences were not statistically significantly different. The area under the curve of glycerol Ra during epinephrine infusion at 84 hours of fasting was significantly lower in the hypertensive than in the normotensive subjects (155 + 39 v 313 _+ 135 gmol/kg/60 rain, respectively, P < .01). DISCUSSION

The results of this study demonstrate that hypertension in obese persons is associated with a downregulation of wholebody lipolytic activity. We used glycerol Ra in plasma as a measure of in vivo lipolytic activity. Basal lipolytic rates and lipolytic sensitivity to catecholamine infusion after 12 and 84 hours of fasting were lower in obese hypertensive than in obese normotensive subjects. Furthermore, blunted lipolyfic rates in hypertensive subjects were associated with a shift in the use of oxidative fuels from lipids to carbohydrates. Therefore, the alteration in adipose tissue lipolytic activity may directly affect substrate oxidation. Several mechanisms may explain the alterations in lipid kinetics observed in our obese hypertensive subjects. One possible factor is the downregulation of [3-adrenergic activity caused by chronic sympathetic nervous system stimulation. We have found that repetitive catecholamine infusion in both lean and obese normotensive subjects causes changes in lipid metabolism similar to those observed in hypertensive subjects of the present study. 11 Intermittent epinephrine infusion three times per day for 6 days increased plasma insulin concentration, decreased whole-body lipolytic rates, decreased the lipolytic response to epinephrine infusion, and shifted substrate oxidation from lipids to carbohydrates. Hypertension in obese subjects is accompanied by increased sympathetic nervous system activity, as manifested by a faster heart rate and increased plasma catecholamine concentrations and urinary catecholamine excrefion,8,9,24,25 compared with obese normotensive subjects. Our hypertensive obese subjects also had higher heart rates and plasma catecholamine concentrations than our normotensive subjects. Differences in plasma insulin concentrations between the normotensive and hypertensive groups also may have contributed to the differences in lipid kinetics. After both 12 hours and 84 hours of fasting, basal plasma insulin concentration in the hypertensive group was higher than that of the normotensive

group. Previous studies have demonstrated that obese hypertensive persons have higher plasma insulin concentrations and insulin resistance (with respect to carbohydrate metabolism) than age- and weight-matched normotensive subjects. 26 However, an increase in plasma insulin without a concomitant resistance to the antilipolytic effect of insulin would have been necessary to inhibit whole-body lipolysis in our hypertensive group. Ferrannini et al27 found that insulin resistance may indeed be specific for certain tissues in hypertensive subjects, because whereas they observed insulin resistance to carbohydrate metabolism in skeletal muscle, insulin sensitivity in adipose tissue was normal in their subjects. The mechanism responsible for increased plasma insulin concentrations and insulin resistance in hypertensive subjects may be related to increased sympathetic nervous system activity. An interaction between the sympathetic nervous system and insulin resistance has been demonstrated in both lean and obese hypertensive subjects. &9,aS,29 This methodology underestimates whole-body lipolytic rates because it quantifies only the rate of glycerol appearance in the systemic circulation; it cannot detect most of the glycerol relased by lipolysis of intraperitoneal fat because of efficient glycerol clearance by the liver. However, it is unlikely that this limitation in methodology affected our conclusions, because intraperitoneal fat represents a small portion (10%) of total body fat mass 3° and the absolute differences in total intraabdominal fat mass between groups was undoubtedly small. In summary, the current study demonstrates a decrease in both basal lipolytic rates and the lipolytic response to epinephrine infusion in obese hypertensive compared with obese normotensive subjects after 12 and 84 hours of fasting. The mechanism(s) responsible for these changes are not clear, but may be related to higher insulin concentrations, downregulation of [3-adrenergic receptors, or a combination of these processes in hypertensive subjects. The alterations in lipid metabolism observed in the present study may be clinically important in therapeutic attempts to decrease body fat by diet or exercise in obese persons with hypertension. ACKNOWLEDGMENT

The authors greatly appreciate the nursing staff of the Clinical Research Center for their help in performing the experimental protocols, LeAnne Romano for technical assistance, and Anita Zinna for preparation of the manuscript.

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