1 Clinical Diabetes and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona 85016; and 2 Laboratory of Human Behavior and Metabolism, Rockefeller University, New York, New York 10021
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ABSTRACT |
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A high 24-h respiratory quotient (RQ), i.e., low
fat oxidation, predicts weight gain. To determine whether impaired fat
mobilization (lipolysis) may contribute to weight gain, we studied the
relation between lipolytic response to nonselective -adrenergic
stimulation and RQ measured in a respiratory chamber in 21 males (11 Caucasians, 10 Pima Indians; age 32 ± 5 yr, weight 93 ± 24 kg,
body fat 30 ± 8%; means ± SD) and 23 females (10 Caucasians,
13 Pima Indians; age 32 ± 9 yr, weight 95 ± 26 kg, body fat 44 ± 8%). Lipolytic response was assessed as the relative increase in
dialysate glycerol concentration (% above baseline) when isoproterenol
(1 µmol/l) was added to the perfusate of a microdialysis probe
inserted in the abdominal subcutaneous adipose tissue. In males, but
not in females, basal RQ measured during sleep from 0500 to 0630 and adjusted for waist circumference was negatively correlated to lipolytic
response (r =
0.66,
P = 0.001). The results suggest that
in males, impaired
-adrenergic-mediated lipolysis may contribute to
low rates of fat oxidation, a condition known to predispose to weight
gain.
indirect calorimetry; microdialysis
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INTRODUCTION |
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THE RELATIVE AMOUNTS of the macronutrients oxidized by
an individual are reflected in the respiratory quotient (RQ), i.e., the
molar ratio between CO2 produced
and O2 consumed. A high RQ, indicating a relatively low fat oxidation, predisposes to weight gain
in Pima Indians (27) and Caucasians (22). There is evidence that an
individual's RQ is influenced by diet composition, energy balance,
body composition, and genetic factors (27), but the physiological
mechanisms underlying this interindividual variability in RQ are
largely unknown. However, a number of findings indicate that the
sympathetic nervous system is an important regulator of fat oxidation
at rest and during exercise. In one study, 2-wk administration of the
-adrenergic agonist terbutaline increased fat oxidation, whereas the
-adrenergic antagonist propranolol decreased fat oxidation (1).
Among the effects of the sympathetic nervous system is its ability to
stimulate adipocyte lipolysis, the hydrolysis of stored fat into
nonesterified fatty acids (NEFAs) and glycerol. This effect is mediated
through
-adrenoceptors by norepinephrine and epinephrine, the only
endogenous hormones with rapid and pronounced stimulatory effects on
lipolysis in adult humans (2). That lipolytic rate is a determinant of
whole body fat oxidation is suggested by the effect of plasma NEFA
concentration on whole body fat oxidation (3, 9). Evidence indicates
that the lipolytic response to
-adrenergic stimulation is decreased in obese men (7) and that this decrease persists after weight loss (8),
suggesting that a low lipolytic response may be causally involved in
the development of obesity.
The purpose of the present study was to test whether a low rate of fat
oxidation is associated with a low lipolytic response to -adrenergic
stimulation. Lipolytic response to locally administered isoproterenol
(a nonselective
-adrenergic agonist) was measured in situ by
microdialysis, and whole body fat oxidation was measured by indirect
calorimetry.
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METHODS |
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Subjects. Twenty-one males (11 Caucasians, 10 Pima Indians) and 23 females (10 Caucasians, 13 Pima Indians) were studied (Table 1). All subjects were healthy, as determined by medical history, physical examination, and routine blood and urine tests, and none took medications or smoked. None had clinical or biochemical signs of thyroid or other metabolic disease. The study was approved by the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases and the Tribal Council of the Gila River Indian Community, and subjects gave written, informed consent. The subjects were residing in our metabolic research unit for 5-7 days and were fed a weight-maintenance diet (20% protein, 30% fat, 50% carbohydrate). Waist circumference was measured at the level of the umbilicus while the subject was in the supine position. Thigh circumference was measured at the gluteal fold while the subject was standing. Body composition was determined by dual-energy X-ray absorptiometry (DPX-1; Lunar Radiation, Madison, WI) (24). After at least 3 days on the weight-maintenance diet, a 75-g oral glucose tolerance test (26) was performed to exclude subjects with diabetes mellitus or impaired glucose tolerance.
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Microdialysis.
The microdialysis procedure has been described in detail elsewhere (12,
23). The procedure was done after 4 days on the weight-maintenance
diet. At 0700, after an overnight fast, with the subject in the supine
position, the microdialysis probe was inserted into the subcutaneous
adipose tissue of the abdomen ~5 cm lateral of the umbilicus. The
probe had a 30 × 1.2-mm shaft with a 10-mm-long 20-kDa cutoff
membrane (CMA/20; CMA/Microdialysis, Solna, Sweden). No local
anesthesia was used. The probe was perfused with Ringer solution
(Abbott, North Chicago, IL) by a precision pump (CMA/102) at a rate of
5 µl/min. In situ recovery of glycerol at 5 µl/min, determined in
separate experiments by dialysate glycerol concentrations at various
flow rates (5), was similar in five lean and five obese subjects (11.1 ± 2.0 and 10.3 ± 4.4%, respectively). For the determination of
regional blood flow, ethanol was added to the Ringer solution for a
final concentration of 50 mmol/l (11). The dialysate collected during
the first 30 min after the insertion of the probes was discarded.
Thereafter, dialysate was collected in 10-min fractions during a 30-min
baseline and during 40 min while isoproterenol (Abbott) was added to
the perfusate for a final concentration of
10
6 mol/l. The dialysate
samples were aliquoted into two portions, one stored at
70°C
for glycerol analysis and the other stored at +5°C for ethanol
analysis within 24 h.
Indirect calorimetry.
After 4 days on the weight-maintenance diet, the subject spent 23 h
in a respiratory chamber (18). No exercise was allowed in the chamber,
and spontaneous physical activity was monitored by a radar system.
Twenty-four-hour energy expenditure and 24-h RQ were calculated as
previously described (18, 27). Basal RQ was calculated as the ratio
between CO2 produced and
O2 consumed for the 90-min period
from 0500 to 0630. Twenty-four-hour energy balance was calculated by
subtracting 24-h energy expenditure from energy intake during the stay
and was expressed as a percentage of energy expenditure.
Analyses. Plasma glucose concentrations were determined by the glucose oxidase method (Beckman Instruments, Fullerton, CA) and plasma insulin concentrations by radioimmunoassay (Concept 4; ICN, Horsham, PA). Plasma insulin concentrations were log10 transformed to approximate a normal distribution. Dialysate glycerol concentrations were determined by a luminometric assay (13, 17). Baseline dialysate glycerol concentration was calculated as the mean value of three 10-min fractions. Isoproterenol-stimulated dialysate glycerol concentration was calculated as the mean value of the three 10-min fractions from 10 to 40 min because a steady-state concentration was not reached until after 10 min of isoproterenol exposure (data not shown). Dialysate ethanol concentrations were determined with the use of a standard enzymatic assay (6). Ethanol outflow-to-inflow ratios were calculated as (dialysate ethanol concentration)/(perfusate ethanol concentration) (14).
Statistical analyses. SAS 6.08 statistical software was used (SAS Institute, Cary, NC). Group comparisons were done by two-tailed t-tests. Relationships between variables were determined by Pearson's correlation coefficient or, when adjusting for covariates, by Pearson's partial correlation coefficient or by multiple linear regression. Adjusted values for RQ were calculated by adding the mean RQ and the residuals obtained from a linear regression model between RQ and waist circumference. Throughout, P < 0.05 was considered significant. Values are means ± SD.
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RESULTS |
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Microdialysis and indirect calorimetry results are reported by gender
in Table 2.
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RQ vs. lipolytic response in males.
Basal RQ and lipolytic response to isoproterenol tended to be
negatively correlated in all males (r = 0.40, P = 0.07) and were
negatively correlated (r =
0.64, P = 0.004) (Fig.
1,
top) when one outlier for waist
circumference was deleted (waist circumference 165 cm, i.e., >5 SD
from the mean of the remaining males). Basal RQ was also negatively
correlated to waist circumference whether or not the outlier was
included (r =
0.60,
P = 0.004 in all males). By multiple
linear regression, basal RQ was found to be inversely associated with
lipolytic response (P = 0.001)
independent of waist circumference. Lipolytic response and waist
circumference were the only significant determinants of basal RQ, both
being negatively associated with basal RQ and explaining 66% of its variability. There was thus no significant additional effect of percent
body fat, waist-to-thigh ratio, fasting plasma insulin concentration,
age, race, or 24-h energy balance on basal RQ. To illustrate the
contribution of lipolytic response to the variability in basal RQ,
waist-adjusted basal RQ against lipolytic response is shown in Fig. 1,
middle
(r =
0.66,
P = 0.001). Basal RQ was not
correlated to baseline dialysate concentration before or after adjustment for covariates. Twenty-four-hour RQ was not correlated to
lipolytic response or baseline glycerol concentration before or after
adjustment for covariates.
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RQ vs. lipolytic response in females. In females, neither basal RQ nor 24-h RQ was correlated to lipolytic response or baseline glycerol concentration before or after adjustment for covariates. Figure 1, bottom, shows the relationship between basal RQ and lipolytic response in females.
Blood flow. In response to isoproterenol, ethanol outflow-to-inflow ratio decreased in both sexes (Table 2), suggestive of an increase in blood flow, but this decrease did not reach statistical significance. The relationship between basal RQ and lipolytic response was independent of the magnitude of the isoproterenol-induced change in blood flow.
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DISCUSSION |
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The purpose of the present study was to test whether whole body RQ,
reflecting the ratio of carbohydrate to lipid oxidation, is related to
in situ -adrenergically mediated lipolytic response. The results
indicate that in males, but not in females, a low lipolytic response to
-adrenergic stimulation is associated with a high RQ, i.e., a low
rate of fat oxidation.
A high RQ predisposes to weight gain (27, 22). In agreement with these
findings, weight-reduced subjects (postobese) have a decreased capacity
for fat oxidation compared with never-obese subjects (4, 16). The
factors determining an individual's fat oxidation are not well
established. Pharmacological studies suggest that effects of the
sympathetic nervous system mediated by -adrenenoceptors play an
important role in regulating fat oxidation (1). One possible mechanism
by which
-adrenergic stimulation could promote fat oxidation is by
its stimulatory effect on lipolysis (2), since plasma NEFA availability
appears to be a determinant of whole body fat oxidation (3, 9). Obesity
appears to be associated with low lipolytic sensitivity to
-adrenergic agonists, both in vitro (20, 21) and in vivo (7, 8, 10,
15, 25).
The present study was designed to test the hypothesis that lipolytic
sensitivity to -adrenergic stimulation is related to whole body fat
oxidation. Lipolysis was assessed in situ by glycerol concentration in
microdialysis dialysate samples. Measurements were made at baseline and
in response to local administration of the nonspecific
-adrenergic
agonist isoproterenol for 40 min. Isoproterenol was infused at a
relatively high concentration
(10
6 mol/l), which has been
shown to cause maximal or near-maximal rates of lipolysis (5). On a
separate day, RQ was measured during sleep in the postabsorptive state
(basal RQ). The main finding of the study is that basal RQ adjusted for
waist circumference was negatively related to lipolytic response to
isoproterenol in males. Basal RQ and lipolytic response were also
negatively related in males without any adjustments when one extremely
obese outlier was excluded. RQ was not related to lipolytic response in
females. This lack of relationship may be due to the fact that most of
the females were very obese (on average 44% body fat) and/or
due to effects of the menstrual cycle and menopausal status. It is also
possible that the abdominal subcutaneous fat depot is less
representative of whole body lipolytic response to
-adrenergic stimulation in females than it is in males. Unlike basal RQ, 24-h RQ
was not correlated with lipolytic response. This was not entirely surprising, since 24-h RQ is influenced mostly by the diet (composition and amount) and physical activity, whereas basal RQ is more dependent on genetic factors (27).
The reviewed literature suggests that the association in males between
low fat oxidation and low lipolytic response to -adrenergic stimulation is explained by reduced plasma NEFA availability. However,
another mechanism, which does not exclude an effect on plasma NEFAs,
may also contribute to the association. If low lipolytic response is
caused by a generalized impairment in
-adrenoceptor function, this
impairment could contribute to lower fat oxidation by affecting
-adrenergically stimulated NEFA uptake in skeletal muscle.
Concomitant impairments in
-adrenergically stimulated lipolysis and
NEFA uptake in muscle have previously been demonstrated in
reduced-obese subjects (8).
The use of microdialysate concentrations of glycerol as a measure of lipolysis has certain limitations, i.e., incomplete recovery (lower glycerol concentration in the dialysate than in the extracellular fluid) and changes in local blood flow in response to the interventions. Recovery was not determined in each subject and may have varied from one subject to another. However, our methodological studies showed small interindividual differences in recovery and no relation with percent body fat, making differences in recovery an unlikely explanation for the observed correlation in males. As demonstrated by others (5), the ethanol outflow/inflow data suggest an increase in adipose tissue blood flow during the isoproterenol infusion. Because local blood flow transports glycerol away from the tissue and thus competes with the microdialysis probe for available glycerol, putative individual differences in vascular response to isoproterenol could have contributed to the findings. However, because the relationship between basal RQ and lipolytic response was independent of individual isoproterenol-induced changes in blood flow, hemodynamic factors appear unlikely to explain the findings.
Fat cell size was not determined in the present study. In general, fat cell size increases with increasing degree of fatness, and larger fat cells have higher maximally isoproterenol-stimulated lipolysis rates in vitro (19). On the other hand, RQ is decreased with increasing degree of fatness (27). It could therefore be argued that the negative relationship between RQ and lipolytic response may be due to the common dependence of both variables on percent body fat. This argument appears unlikely for two reasons. First, the analysis showed that the relationship between basal RQ and lipolytic response was independent of percent body fat. Second, when measured in vivo, lipolytic response appears decreased in obese individuals (7, 10, 15, 25).
In conclusion, the present study indicates that in males, a low
lipolytic response to isoproterenol in abdominal subcutaneous adipose
tissue is associated with a low rate of fat oxidation, a known risk
factor for body weight gain, suggesting that a low lipolytic response
to -adrenoceptor stimulation may contribute to weight gain.
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ACKNOWLEDGEMENTS |
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The Clinical Diabetes and Nutrition Section is indebted to the members of the Gila River Indian Community, who for more than 30 yr have contributed to studies of the development of non-insulin-dependent diabetes mellitus. We thank the individuals, Pimas and non-Pimas, who volunteered for this study; Joy Truesdale for technical assistance; and the technical, nursing, and dietary staffs of the Clinical Diabetes and Nutrition Section.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence: S. Snitker, National Institutes of Health, 4212 N 16th St., Rm. 541, Phoenix, AZ 85016.
Received 29 January 1998; accepted in final form 21 May 1998.
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