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Original Studies |
Section of Applied Physiology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Dr. Robert C. Hickner, East Carolina University, 371 Ward Sports Medicine Building, Greenville, North Carolina 27858. E-mail: hicknerr{at}mail.ecu.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Simultaneous assessment of regional lipolysis in sc adipose tissue by microdialysis and whole body lipolysis by stable labeled isotope kinetics allows for determination of the relative importance of sc vs. other depots of adipose tissue (presumably intraabdominal or skeletal muscle) in the antilipolytic response to insulin. In this context, the specific aim of this study was to evaluate the effects of 10 days of exercise on the suppression by insulin of whole body [glycerol rate of appearance (Ra)] and regional (microdialysis of sc adipose tissue) lipolysis during a hyperinsulinemic-euglycemic clamp in obese premenopausal women.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Ten obese premenopausal women [>28% body fat; maximal oxygen uptake (VO2max), <35 mL/kg·min] participated in the study after giving their informed consent according to the institutional review board at Washington University School of Medicine. The volunteers were not suffering from any chronic or acute illnesses, and none of the subjects had a prior history of cardiovascular disease. Three of the subjects were glucose intolerant as determined by an oral glucose tolerance test performed as a screening for participation in this study. Subjects reported that they were weight stable for the 6 months preceding the investigation. All subjects were eumennorheic, in that all had monthly menses for the 12 months preceding the investigation.
Protocol
Subjects were studied before and after 10 days of endurance training. Whole body lipolysis was evaluated by 2H5-glycerol kinetics in the basal state and during two low dose insulin infusions. Regional lipolysis was evaluated simultaneously by placing microdialysis probes in sc adipose tissue in the abdominal (two probes) and femoral (two probes) regions.
Endurance training
The endurance training program consisted of 1 h/day (10 days of 14 consecutive days) of exercise on a motorized treadmill, cycle ergometer, stair-climbing ergometer, and/or rowing ergometer. Twenty-five minutes of the exercise session were performed at a constant workload set to elicit approximately 70% VO2max. The final 30 min of the exercise session was designed to consist of five 5-min intervals at an exercise intensity set to elicit 100% VO2max (the actual intensity maintained by subjects was 90% VO2max), with 2.5 min of rest between intervals. This exercise training program has been previously demonstrated to result in significant improvements in VO2max in the absence of measurable changes in body composition. The effects of exercise training are therefore investigated without the confounding factor of changes in body composition.
Body composition and fat distribution
Fat and fat-free masses were measured by dual energy x-ray absorptiometry (QDR-1000/w, Hologic, Inc., Waltham, MA) before and after endurance training, as described previously using version 5.64 of the enhanced whole body analysis program (10). Regional adiposity was also evaluated anthropometrically. Circumference measurements were made at the waist, hip, and midthigh. The anatomical landmarks used to locate the circumference sites in a reproducible manner were those described in the Anthropometric Standardization Reference Manual (11).
VO2max
VO2max was determined on a motorized treadmill. Subjects walked at 3 mph for 3 min, and speed was then increased by 1.0 mph every 3 min to 5 mph. The speed was then held constant, and grade was increased by 2% every minute until exhaustion. The fractional oxygen and carbon dioxide content of expired air were measured continuously, and oxygen uptake was calculated for each 30-s interval using an automated, open circuit system (8). At least two of the following criteria were met to establish that VO2max had been attained: a plateau in VO2 despite an increase in treadmill speed or grade, attainment of age-predicted maximal heart rate, and a respiratory exchange ratio greater than 1.10.
Hyperinsulinemic-euglycemic clamp and glycerol kinetics
The hyperinsulinemic-euglycemic clamp studies were performed as
described previously (12) at the Washington University General Clinical
Research Center after an overnight fast. Although the time of the clamp
procedure was randomized with respect to the menstrual cycle for each
subject, subjects were studied at the same point in the menstrual cycle
before and after 10 days of exercise. This was accomplished by delaying
the beginning of exercise training for 16 days after the initial
(pretraining) hyperinsulinemic-euglycemic clamp. In the trained state,
subjects were studied 16–24 h after their last exercise bout to study
individuals in the habitual state of exercise training. Subjects were
instructed to consume at least 250 g carbohydrate/day for the 3
days before the clamp procedure. Three baseline venous blood samples
were obtained at 5-min intervals to determine plasma substrate and
hormone concentrations and background isotope enrichments. A primed
(1.5 µmol/kg), constant (
0.1 µmol/kg·min) infusion of
2H5-glycerol (99%; Tracer
Technologies, Newton, MA) was started and continued for 270 min (90-min
basal period, 90 min at each insulin infusion rate). The actual isotope
delivery rate was determined by measuring the enrichment of the isotope
in the infusate. The 90-min basal period was followed by two sequential
90-min primed, constant insulin infusions. Insulin was delivered at
rates of 10 and 20 mU/m2·min. It was
anticipated that these infusion rates would result in plasma insulin
concentrations of approximately 15 and 35 µU/mL. These levels were
chosen because the insulin concentration for a half-maximal suppression
of lipolysis has been shown to be approximately 35 µU/mL in obese
subjects (13).
The rate of glucose disposal was estimated from the steady state glucose infusion rate over the final 30 min of each insulin infusion. It is recognized that this may have resulted in an underestimate of the actual glucose disposal rate, as hepatic glucose production may not have been completely suppressed at the low insulin concentrations. Furthermore, the insulin infusion rates were expected to result in plasma insulin concentrations in the low, flat portion of the sigmoidal dose-response curve for the effect of insulin on glucose disposal (i.e. very small changes in glucose disposal could be expected). The glucoregulatory data were therefore not the focus of these investigations.
Blood samples were obtained at 10-min intervals during the final 30 min of each of the 90-min stages for the determination of plasma substrate and hormone concentrations as well as isotope enrichments. Plasma samples were stored at -80 C and subsequently analyzed for free fatty acids (NEFA kit, Wako Chemicals, Dallas, TX), glycerol (14), insulin (15), and epinephrine and norepinephrine (16). Interassay coefficients of variation are 8% for glycerol, 14% for insulin, 4% for free fatty acids, and 11% for catecholamines in the concentration range of samples in this study.
2H5-glycerol analysis
The analysis of 2H5-glycerol was performed, as we have previously described (6), using a modification of the negative ion chemical ionization gas chromatography-mass spectrometry method described by Gilker et al. (17). The Ra of glycerol over the last 30 min of each stage was calculated using the nonsteady state equations of Steele (18), assuming a volume of distribution for glycerol of 270 mL/kg (19). The coefficient of variation for Ra was 7.1 ± 2.2% as determined from day to day reproducibility of enrichment determinations (n = 6).
Assessment of regional lipolysis by microdialysis
The microdialysis probes (DL-3, Bioanalytical Systems, Inc., West Lafayette, IN) consisted of inlet tubing (30 cm) and outlet tubing (15 cm) separated by a polyacrylonitrile dialysis membrane (3 cm in length; id, 0.25 mm; od, 0.35 mm). Probes were sterilized using ethylene oxide gas and were soaked for a minimum of 16 h overnight in 5% ethanol while perfused with Ringer’s solution to remove the glycerol coating that was placed on the dialysis membrane during the manufacturing process. The absence of measurable glycerol on the microdialysis membrane was verified by an absence of glycerol in the dialysate during perfusion of the microdialysis probes with Ringer’s solution in vitro before insertion into the research subjects.
Microdialysis probes were inserted under sterile technique and local
anesthesia (0.1 mL 1% lidocaine without epinephrine) into the
abdominal (two probes, bilaterally 3 cm lateral to the umbilicus)
and femoral (two probes, 3 cm apart, midthigh) sc adipose tissue in
each subject. This was accomplished by inserting a 14-gauge catheter
through the skin, advancing it through the sc adipose tissue parallel
to the skin, and then exiting through the skin. The needle was
withdrawn, the microdialysis probe was threaded through the catheter,
and the catheter was removed, leaving the membrane portion of the probe
embedded in the adipose tissue.
Microdialysis probes were perfused (Harvard infusion pump, model 22, Harvard Apparatus, South Natick, MA) at 2.0 µL/min with Ringer’s solution containing 2.5 mmol/L glucose and 5 mmol/L ethanol for 60 min. No samples were collected during this time to allow for equilibration of the microdialysis system and to allow the initial trauma of probe insertion to subside (20, 21). Glucose (2.5 mmol/L) was added to the perfusate to minimize the drainage of glucose from the interstitial fluid. No significant net loss or gain of perfusate fluid occurred over the dialysis membrane during the microdialysis process under the conditions of this experiment, as verified by weighing of dialysate samples. The inclusion of ethanol in the perfusate permitted the detection of changes in blood flow in the region surrounding the probe (22, 23). Ethanol at the concentration used (5 mmol/L) has been shown to have no effect on lipolysis in sc adipose tissue (23). The ethanol data are presented as the outflow/inflow ratio ([ethanol]dialysate/[ethanol]perfusate), which is inversely related to blood flow in the region surrounding the microdialysis probe (21). The outgoing dialysate was collected in 15-min fractions (30 µL). The dialysate samples were stored at 4 C and analyzed within 48 h for glycerol (14) and ethanol (21). We have previously determined the relative recovery for glycerol over this type of dialysis membrane to be approximately 60% in group of premenopausal women (6). The range of recoveries, however, can be from 50–70% at the perfusion flow rate and membrane characteristics employed, and can vary with changes in adiposity (24, 25).
Statistics
Differences were analyzed by one-way repeated measures ANOVA. If significance was obtained, the Newman-Keuls post-hoc analysis was used to identify group differences. The effects of insulin infusion on blood or dialysate parameters as well as differences between pre- and posttraining VO2max were evaluated using two-tailed paired Student’s t tests. All data are expressed as the mean ± SE. The level of significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subject characteristics with respect to age,
VO2max, body composition, and fat distribution
are presented in Table 1
. Body weight and
indexes of fatness were unchanged after training, whereas
VO2max (milliliters per kg/min) was increased
6.4% (P < 0.05) after training.
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There were no significant differences between pre- and
posttraining in basal serum free fatty acid, epinephrine,
norepinephrine, and insulin concentrations (Table 2). No changes from basal were measured
in norepinephrine and epinephrine concentrations. Insulin
concentrations increased to a similar extent upon insulin infusion
before and after training. Free fatty acid concentrations were not
suppressed to a greater extent than the low dose during the moderate
dose insulin infusion after, compared to before, training.
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Basal. In the basal state, plasma glycerol concentrations were higher after training (100.3 ± 19.8 µmol/L) than before training (86.7 ± 17.0 µmol/L; P < 0.05). Basal abdominal and femoral dialysate glycerol concentrations were not different after, compared to before, training.
Glycerol Ra values were 2.70 ± 0.36 and 2.69 ± 0.30 µmol/kg BW·min under basal conditions before and after training, respectively (P = NS). Body weight and fat mass did not change with training; glycerol Ra values per unit fat mass were therefore also similar before and after training.
Insulin infusion. Plasma and dialysate glycerol concentrations
were significantly reduced both before and after training in response
to the first insulin infusion (Fig. 1).
The only further significant reduction in glycerol concentration during
the second insulin infusion before training occurred in the femoral
dialysate. After training, glycerol Ra and glycerol concentrations in
plasma and dialysates were reduced during the moderate dose compared to
the low dose insulin infusion.
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). Glycerol Ra was reduced to a greater extent after
training compared to before training only during the moderate dose
insulin infusion. There was no greater reduction in dialysate glycerol
concentration after training compared to before training in either
abdominal or femoral sites.
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The basal ethanol outflow/inflow ratios from probes in abdominal sc adipose tissue were 0.537 ± 0.028 and decreased to 0.507 ± 0.033 (P < 0.01) during the low dose insulin infusion. The outflow/inflow ratios were further reduced to 0.475 ± 0.032 (P < 0.01) during the second insulin infusion. Basal ethanol outflow/inflow ratios from probes in femoral sc adipose tissue were 0.561 ± 0.028, were reduced to 0.490 ± 0.028 (P < 0.01) during the low dose insulin infusion, and were further reduced to 0.440 ± 0.031 during the moderate dose insulin infusion (P < 0.01). These data indicate that nutritive blood flow per tissue volume was increased upon insulin infusion.
Although, as noted above, ethanol outflow/inflow ratio decreased (increase in adipose tissue nutritive blood flow) in response to insulin infusion before endurance training, similar decreases in ethanol outflow/inflow ratio were recorded after training. Ethanol outflow/inflow ratios from probes placed in abdominal adipose tissue decreased from 0.564 ± 0.074 before insulin infusion (P = NS compared to pretraining) to 0.485 ± 0.044 (P < 0.01) and 0.459 ± 0.041 (P < 0.01) during the low and moderate dose insulin infusions, respectively. Ethanol outflow/inflow ratios from probes placed in femoral adipose tissue decreased from 0.585 ± 0.041 before insulin infusion (P = NS compared to pretraining) to 0.499 ± 0.036 (P < 0.01) and 0.447 ± 0.028 (P < 0.01) during the low and moderate dose insulin infusions, respectively.
Glucose disposal
During the first insulin infusion, glucose disposal rates were lower after training (1.69 ± 0.28 mg/kg·min) than before training (1.98 ± 0.32 mg/kg·min; P < 0.05). During the second insulin infusion, glucose disposal rates tended to be higher after training (4.05 ± 0.72 mg/kg·min) than before training (3.76 ± 0.69 mg/kg·min; P = 0.1).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Regional differences in the antilipolytic effect of insulin before training
With respect to regional differences in response to the antilipolytic action of insulin, the data from the present study support our previous findings (26, 27) that femoral sc adipose tissue is more resistant to the effects of insulin than is abdominal sc adipose tissue. Femoral adipose tissue was more resistant to the antilipolytic action of insulin than was abdominal adipose tissue, which was more resistant than the whole body antilipolytic response to insulin, both before and after training. These findings coupled with previous observations that femoral adipocytes of women have a metabolic profile that favors storage to a greater degree than abdominal adipocytes (28) indicate that triglyceride turnover is higher in femoral than abdominal sc adipocytes. Our reports of a decreased antilipolytic action of insulin in femoral compared to abdominal sc fat depots are not consistent with in vitro findings of Bolinder et al. (29) and Arner et al. (30) or in vivo findings of Jensen et al. (7). These researchers report equal or greater antilipolytic response in femoral than abdominal adipocytes.
Effect of training on basal glycerol Ra and dialysate glycerol concentrations
Basal glycerol Ra was unchanged after training, although fasting glycerol concentrations were elevated. Basal glycerol Ra and fasting plasma glycerol concentrations were more than 50% higher in the trained group than in the sedentary groups in our previous cross-sectional study of trained and sedentary women (6). Although the elevated fasting plasma glycerol concentrations may (31) or may not (32) be due to an acute effect of the recent exercise bout (16–24 h before testing), the elevated glycerol Ra appears to be due to a cumulative training effect, which cannot be duplicated with only 2 weeks of endurance training. In our previous study of premenopausal women, differences were also found between trained and sedentary individuals in dialysate glycerol concentrations from probes placed in abdominal sc adipose tissue under basal conditions. These differences were proportional to differences in Ra per kg fat mass. It was therefore suggested that the slightly higher lipolytic rate in sc adipose tissue of trained compared to sedentary nonobese individuals may have accounted for the increased basal whole body lipolysis in the trained group (glycerol Ra). This effect of training on basal glycerol release from sc adipose tissue also appears to be an effect of an extended period of endurance training and was not seen in the present study after 2 weeks of endurance training.
Effect of training on the antilipolytic response to insulin: site of action
Although there is limited information regarding the effects of exercise training on the antilipolytic response to insulin, Suda et al. (33) reported an increased suppression of lipolysis by insulin in the epidydimal fat pad of exercise-trained rats. We have now demonstrated an increased suppression of lipolysis by insulin at the whole body level, but not in sc adipose tissue, in exercise-trained humans. The negative results of our previous study (6) may have been due to the small sample size (n = 6) and the cross-sectional study design. However, trained and sedentary premenopausal women in the previous study were nonobese and were therefore relatively insulin sensitive compared to the current group of premenopausal women, which was comprised of mildly to morbidly obese women. A training effect with regard to the antilipolytic action of insulin may be more predominant, and therefore easier to detect, in insulin-resistant individuals.
Although intraabdominal fat mass was not determined before and after training in the present study, it is unlikely that intraabdominal fat mass changed significantly after only 2 weeks of training in the absence of changes in body composition. Furthermore, the hormonal milieu (epinephrine, norepinephrine, insulin) was not different after, compared to before, training. If intraabdominal adipose tissue were the site of increased suppression of lipolysis after training, increased insulin action must have been the mechanism. It has been shown that resistance to the antilipolytic action of insulin in vitro is more pronounced in adipocytes from the omental adipose depot than in those from sc adipose depots (34). The antilipolytic response to insulin in intraabdominal adipose depots may therefore have been enhanced to a greater extent than in sc adipose depots after successive days of exercise.
Another candidate site for training effects on the anti-lipolytic response to insulin would be im triglyceride stores within or between muscle fibers. Studies of glycerol release in men using arteriovenous differences over the limb (leg) have been inconclusive (3, 4, 35). Dela et al. showed both no change in (4, 35) as well as an increased response to (3) the antilipolytic action of a maximal dose of insulin after 10 weeks of a one-legged exercise training program. It is possible that there is a response to training in skeletal muscle that does not occur in adipose tissue. There is, in fact, evidence that different phosphodiesterase subtypes regulate antilipolysis in skeletal muscle and adipose tissue (23).
Microdialysis probes were not placed in skeletal muscle or intraabdominal fat of the subjects in the present study. Whether im or intraabdominal triglyceride stores were the site of increased antilipolytic insulin action after training must therefore be addressed in future studies; investigation of the latter is clearly limited due to the inaccessibility of intraabdominal fat.
Potential limitations
In the present investigations, the dialysate glycerol concentration was not a direct measure of the interstitial glycerol concentration due to incomplete equilibration (recovery) of glycerol over the dialysis membrane. Changes in nutritive blood flow could potentially result in changes in dialysate glycerol concentration independent of changes in the lipolytic state of the fat cells (23, 36, 37). Microdialysis was used in the present study to monitor both lipolysis and nutritive blood flow in sc adipose tissue. Ethanol outflow/inflow ratio was reduced (increased nutritive blood flow) in both adipose tissue depots in response to insulin infusion. Although there were no significant differences in ethanol outflow/inflow ratios under basal and insulin-stimulated conditions, the mean outflow/inflow ratio in the abdominal site appeared to be higher at baseline and was reduced in response to insulin infusion to a greater extent after, compared to before, training. This would indicate that the nutritive blood flow response to insulin in the abdominal adipose tissue depot might have been greater after, compared to before, training, thus resulting in greater removal of glycerol from the interstitium. The percent decline in the abdominal dialysate glycerol concentration after training may therefore have been exaggerated. Consequently, the data for the percent decline in dialysate glycerol concentration before and after training are probably more similar than they appear. Alterations in sc adipose tissue nutritive blood flow, therefore, did not change the main conclusion regarding dialysate glycerol results, e.g. there were no differences in the antilipolytic response to insulin in sc adipose tissue after, compared to before, endurance training.
Summary
Before training, the greatest antilipolytic effect of insulin was seen in the plasma glycerol concentrations, whereas the poorest antilipolytic response to insulin was seen in the femoral sc adipose tissue glycerol levels. Thus, the order of the antilipolytic response (determined by dialysate glycerol concentrations) to insulin was plasma > abdominal sc adipose tissue > femoral sc adipose tissue. There was no effect of training on this order of sensitivity, although there were increases in the magnitude of the whole body antilipolytic response. During insulin infusion, there was a greater relative suppression of whole body lipolysis after, compared to before, endurance training. However, there was no effect of training on the relative degree of suppression of regional sc adipose tissue lipolysis. Basal lipolysis per kg fat mass was not affected by 2 weeks of endurance training. These data indicate that improvements in the antilipolytic action of insulin can be made in the absence of significant improvements in the sc adipose tissue response to insulin. Fat depots responding to training may be intraabdominal fat, although an improvement in the antilipolytic action of insulin on fat in muscle (i.e. between or within muscle fibers) could not be ruled out. These findings demonstrate that obese women can improve the antilipolytic response to insulin through endurance training.
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Acknowledgments |
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Footnotes |
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2 Initially supported by Institutional National Research Service
Award AG-00078 and later by Individual National Research Service Award
DK-09542.
3 Supported by NIH Research Career Development Award AG-00663.
Present address: University of Colorado Health Sciences Center, 4200
East Ninth Avenue, Box B-179, Denver, Colorado 80237.
Received August 31, 1999.
Revised December 15, 1999.
Accepted January 4, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-2 adrenoceptors in regulating lipid
mobilization from human adipose tissue. J Clin Invest. 91:1997–2003.
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) and
after (
)
2 weeks of endurance training. Data are expressed as the mean ±
SE (n = 10). *, Different from basal
(P < 0.05). **, Different from pretraining
(P < 0.05).
) 2 weeks of endurance
training during a low dose (10 mU/m2·min) and moderate
dose (20 mU/m2·min) insulin infusion. Data are the
mean ± SE (n = 10) and are expressed as a
percentage of basal. **, Different from pretraining
(P < 0.05). +, Different from low dose
(P < 0.05).