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Methods |
Department of Physical Education and Sport Science, Aristotle University of Thessaloniki, Thessaloniki, Greece
DOI 10.1194/jlr.C200003-JLR200
1 To whom correspondence should be addressed. e-mail: mougios{at}phed.auth.gr
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ABSTRACT |
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In conclusion, our data show that TG lipase activity in human adipose tissue peaks early during exercise and subsequently decreases despite the maintenance of the physical stimulus.
Abbreviations: HSL, hormone-sensitive lipase
Supplementary key words lipolysis • hormone-sensitive lipase • phosphorylation • assay • biopsy
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INTRODUCTION |
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TG lipase activity of adipose tissue is usually measured against a synthetic substrate, 1(3)-[3H]oleoyl-2-O-oleylglycerol, because its rate of hydrolysis is 10 times that of TG and independent of the phosphorylation state of the enzyme (10). The latter feature, however, precludes the use of the synthetic substrate to determine the actual activity of the enzyme and to monitor acute changes in response to endogenous or exogenous stimuli. Therefore, to study the effect of exercise on TG lipase activity, an assay sensitive to the phosphorylation state of the enzyme is needed.
The aim of the present study was to investigate the effect of moderate exercise on TG lipase activity in human adipose tissue. To achieve this, we have developed a novel assay of the actual activity of the enzyme against endogenous TG.
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METHODS |
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Experimental protocol
Each subject was transported to the laboratory by car on a separate day at 9 AM, after an overnight fast and rest. Following 15 min of rest, an adipose tissue sample was obtained by needle biopsy from the buttock area without anesthesia, as described (11). Subsequently, 10 of the subjects exercised on a Kettler KX1 bicycle ergometer (Ense-Parsit, Germany) for 30 min at a power output corresponding to a heart rate of 120–130 beats min-1, being monitored continuously by a Polar Accurex monitor (Kempele, Finland). The power output had been determined during a preliminary test performed at least 1 week earlier. Adipose tissue biopsy was performed at 5, 15, and 30 min of exercise, as well as after 15 min of passive recovery. All samples were taken with the subjects seated on the bicycle and with a 5- to 10-s interruption of cycling at 5 and 15 min of exercise. The other five subjects were sequentially biopsied as above, the only difference being that they rested throughout the sampling period.
TG lipase assay
The determination of TG lipase activity was performed immediately after biopsy, without storage of the adipose tissue sample. The sample was transferred into a glass homogenizer (Kontes, Vineland, NJ) containing 300 µl of a potassium phosphate buffer (0.1 mol/l, pH 7.0), to which the following substances (all from Sigma, St. Louis, MO) had been added in order to prevent protein dephosphorylation, phosphorylation, and degradation: EDTA (1 mmol/l), "phosphatase inhibitor cocktail I" (containing microcystin LR, cantharidin, and (-)-p-bromotetramisole in DMSO) in 100-fold dilution, inhibitor of the cAMP-dependent protein kinase (rabbit sequence, 2.5 µmol/l), antipain (20 mg/l), leupeptin (20 mg/l), and pepstatin (10 mg/l). The sample was weighed and homogenized with approximately 20 manual strokes, while buffer was being added to a final volume of 20 times the weight of the tissue. The total time from sampling to complete homogenization was approximately 30 s. The homogenate was then transferred to a test tube and was incubated at 37°C under continuous stirring. At 0 and 30 min, 10 µl aliquots were removed in triplicate from the homogenate and were transferred into 1 ml of 2-propanol-heptane-1 N H2SO4, 40:10:1 (v/v/v), to denature TG lipase. This solution also contained 70 µg of triheptadecanoyl glycerol (Sigma) as the internal standard for the subsequent chromatographic analysis.
Lipids were extracted from the above solution as described (12) and were separated by TLC on silica gel plates with petroleum ether-diethyl ether-acetic acid, 80:20:1 (v/v/v), as a developer. Lipid spots were located under ultraviolet light after spraying with a solution of dichlorofluorescein in ethanol and the spot corresponding to TG was excised and incubated in 0.5 ml of methanolic sodium methoxide (Sigma) at 50°C for 10 min (13). The fatty acid methyl esters thus produced were extracted with hexane and were separated by gas chromatography as described (14). Methyl esters were quantified by comparing the area under their peaks in the chromatogram to that of methyl heptadecanoate (produced by transmethylation of the internal standard). The µmol of TG in each aliquot of the homogenate were calculated as the sum of the µmol of the methyl esters divided by 3. TG lipase activity was then calculated as the rate of TG decrease over the incubation period. The coefficient of variation of the assay was 12%.
Statistical analysis
Results are reported as the mean ± SE. Significant changes in the percentage of each TG acyl group from 0 to 30 min of incubation were detected by performing paired Student's t-tests. Comparisons of TG lipase activity between exercise and rest, as well as among the five time points of adipose tissue sampling were performed by two-way (condition x time) ANOVA with repeated measures on time. Post-hoc pairwise comparisons were performed through simple main effects analysis. The level of statistical significance was set at 
= 0.05. The SPSS (version 10.0) was used for all analyses.
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RESULTS |
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Seven fatty acid methyl esters derived from the TG of adipose tissue were detected in considerable amounts by gas chromatography, namely myristate (14:0), palmitate (16:0), palmitoleate (16:1
7), stearate (18:0), oleate (18:19), vaccenate (18:17), and linoleate (18:26). Their molar percentage distribution at the onset of incubation is presented in Table 1. The distribution after 30 min of incubation (not shown) was very similar to that at the onset.
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DISCUSSION |
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To preserve, throughout the incubation period, the phosphorylation state of TG lipase at the time of adipose tissue sampling and the integrity of the enzyme, the homogenization buffer contained: i) EDTA to chelate Ca2+ and Mg2+. Ca2+ is a known activator of proteinases, whereas Mg2+ is required by enzymes utilizing ATP, such as protein kinases and acyl-coenzyme A synthetases (involved in TG resynthesis), as well as by protein phosphatase-2C (17, 18), which, along with protein phosphatases-1 and 2A, has been shown to dephosphorylate TG lipase (18); ii) a phosphatase inhibitor mixture; iii) an inhibitor of the cAMP-dependent protein kinase to prevent additional phosphorylation of TG lipase; iv) three proteinase inhibitors (antipain, leupeptin, and pepstatin).
An additional advantage of our assay is the use of mild homogenization so as to conserve the morphology of the intracellular lipid droplets. This is important because TG hydrolysis requires the translocation of the lipase from the cytosol to the surface of the droplets (19, 20). Adhesion of the lipase to the droplets is modulated by proteins, such as perilipins. The latter are located at the droplet surface and, in response to lipolytic stimuli, undergo conformational changes facilitating the interaction of the lipase with TG (21, 22). Therefore, it was crucial to preserve all factors affecting TG lipase activity in a state as close to the in vivo condition as possible.
To document the reproducibility of our assay and to control a possible carry-over effect of sequential biopsying on TG lipase activity, we have included a control group who rested throughout the sampling period. Our data suggest that the assay was reproducible and that no carry-over effect occurred.
The main finding of the present study was that TG lipase activity increased early during exercise, reaching a peak at 5 min, which was over 6-fold higher than baseline. It is interesting that, although the subjects continued to exercise, lipase activity gradually decreased and was significantly lower at all time points compared with 5 min. In accordance with this finding, TG lipase activity in rat muscle was found to be significantly higher than baseline after 1 and 5 min, but not after 10 or 60 min of electrical stimulation (23). This decrease in lipase activity despite the maintenance of the physical stimulus may be attributed to the well-known desensitization of ß-adrenergic receptors, which would result in decreased activation of the cAMP cascade and, hence, decreased activation of TG lipase. Indeed, it has been reported that ß-adrenergic stimulation of lipolysis in human adipose tissue was significantly higher before exercise than after 60 min of moderate exercise (24).
The absence of significant changes in the percentage distribution of TG acyl groups during the assay suggests that there was no preferential hydrolysis of some acyl group(s) by the lipase. This finding contrasts with published data that show a preferential release of polyunsaturated fatty acids from synthetic TG-analogs by crude preparations of rat HSL (25) and from isolated human adipocytes stimulated by lipolytic agents (26). The reasons for the discrepancy between the results of the present study and the aforementioned ones may be the shorter incubation time (0.5 vs. 1.5–24 h) or the different experimental conditions.
In conclusion, the present study demonstrates that TG lipase activity in adipose tissue peaks at 5 min of exercise and decreases afterwards, although the exercise stimulus continues. To measure acute changes in lipase activity, a novel assay, sensitive to the phosphorylation state of the enzyme, was developed. This assay may be further used to study the effect of different exercise conditions on TG lipase activity. Additionally, it would be interesting to examine the effect of exercise on the activity of the enzyme in different population groups, such as the obese, females, and athletes. Finally, our assay permits research on the effect of pharmacological or dietary interventions on TG lipase activity aimed at understanding the mechanism of action of known lipolytic agents and the development of new ones, given the intense interest of modern societies in discovering effective ways to reduce body weight and, in particular, body fat.
Manuscript received February 14, 2002 and in revised form April 26, 2002.
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