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Original Studies |
Departments of Medicine and Surgery (A.T.), Huddinge Hospital, Karolinska Institute, 14186 Huddinge, Sweden; and INSERM U-317, Institut Louis Bugnard, Faculté de Médecine, Hôpital Rangueil (M.D., D.L.), 31403 Toulouse, France
Address all correspondence and requests for reprints to: Signy Reynisdottir, M.D., Ph.D., MK Division, Huddinge Hospital, S-141 86 Huddinge, Sweden.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In humans, catecholamines are the most potent activators of lipolysis,
the breakdown of triglycerides into glycerol and FFA. They modulate
adenylyl cyclase activity via stimulatory ß1-,
ß2-, and ß3-adrenoceptors and inhibitory
2-adrenoceptors (5). When the ß-adrenergic pathway is
predominant, the intracellular level of cAMP increases activating the
cAMP-dependent protein kinase. The kinase phosphorylates and activates
hormone-sensitive lipase (HSL) (6, 7). HSL catalyzes the breakdown of
triglycerides and diglycerides, which is the rate-limiting step of
adipose tissue lipolysis. Regional differences in HSL activity have
recently been described in rats (8, 9), but have not previously been
examined in man.
In the present study the possible role of HSL in determining regional differences in the lipolysis rate was investigated in human fat cells from omental and sc abdominal adipose tissue.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The study included 21 subjects (9 men and 12 women) who were recruited among otherwise healthy patients undergoing elective open or laparoscopic surgery because of either gallstones or obesity. To examine the role of fat cell size, the subjects were selected to represent a wide range in body weight, but there was no selection on the basis of age or sex. None had jaundice, and all were free from medication. For methodological studies small fragments of adipose tissue from an additional 22 subjects were obtained during surgery. The study was explained in detail to each participant, and consent was obtained. The study was approved by the ethics committee at the Karolinska Institute.
The subjects fasted overnight, and only saline was infused iv before
the fat biopsy. The surgical procedures started at 0800 h. General
anesthesia was induced by a short acting barbiturate and maintained by
phentanyl and nitrous oxide. Fat biopsies (
1–3 g) were removed from
both the abdominal sc and the omental adipose tissue of each subject
within 30 min after the start of general anesthesia. The tissue was
immediately transported to the laboratory in saline.
Isolation of fat cells and determination of fat cell number
This procedure has been described in detail previously (10). Adipose tissue was cut into small fragments (10–20 mg), and isolated fat cells were prepared by collagenase treatment (11). The cells were kept in a Krebs-Ringer phosphate buffer with albumin. The cells were packed through centrifugation at 200 rpm for 1 min. Aliquots of 300 and 200 µL were frozen directly into liquid nitrogen for later analysis of HSL activity and messenger ribonucleic acid (mRNA) expression, respectively. Direct microscopic determination of the fat cell diameter was performed according to the method of DiGirolamo and co-workers (12). An aliquot of the fat cell suspension was placed on a glass slide and examined with a Zeiss microscope (Zeiss, New York, NY) equipped with a caliper scale. The diameters of 100 cells were measured. Mean cell volume, surface area, and weight of the fat cells were calculated, taking into consideration the skewness in the distribution of the cell diameter (13).
The number of fat cells incubated in the lipolysis experiments was determined as follows. The lipids in an aliquot of the incubation mixture were extracted according to the method of Dole and Meinertz (14), and the lipid content was determined gravimetrically. The number of fat cells incubated was obtained by dividing the mean lipid content of the incubation tubes by the mean fat cell weight, assuming that lipids constitute more than 95% of the fat cell weight.
Two ways of determining the number of fat cells homogenized for
analysis of HSL activity were compared in separate methodological
experiments on small samples of both omental and sc adipose tissue from
32 subjects. First, the number of cells was calculated, as described
above, by extraction of the lipids from a 25-µL aliquot of packed fat
cells. Second, the number of fat cells was estimated by dividing the
amount of packed cells used in the HSL assay (300 µL) by the mean fat
cell volume. In simple regression analysis (Fig. 1
), there was a close correlation between
these two methods in both adipose regions examined (omental: r =
0.94; P = 0.0001; sc: r = 0.92; P
= 0.0001), and analysis of covariance did not reveal any significant
difference between the two regression lines. Although the latter method
gave a higher estimate of the cell number by 24 ± 2% in the sc
and by 26 ± 3% in the omental fat samples, respectively, the
slope of the regression line did not differ significantly from 1 in
either region, indicating only a minor effect of variations in cell
size and the procedure of packing the fat cells on this relationship.
The slopes were 1.1 and 1.3 in omental and sc regions, respectively. As
the amount of adipose tissue that can be obtained during laparoscopic
surgery is frequently scarce, the latter mode of determination was
subsequently used.
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This assay has previously been described in detail (10). It was performed in 19 of the 21 subjects. A diluted suspension of fat cells was incubated at 37 C in duplicate samples, with or without increasing concentrations (10-3–10-9) of the following lipolytic agents, acting at different levels of the lipolytic cascade: the nonselective ß-adrenergic agonist isoprenaline; forskolin, which stimulates adenylyl cyclase; and dibutyryl cAMP (dcAMP), a phosphodiesterase-resistant cAMP analog that activates cAMP-dependent protein kinase. Glycerol release to the incubation medium was used as an index of the lipolysis rate. After 2-h incubation, an aliquot of the incubation medium was removed for analysis of glycerol content using an automated bioluminescence assay (15).
All of the agonists caused a dose-dependent increase in glycerol release, reaching a plateau at the highest agonist concentrations. The responsiveness to each agonist was determined as the maximum stimulated lipolysis rate. The lipolysis rates in the presence or absence of agonist were related to either lipid weight or the number of incubated cells.
Assay of HSL activity
This assay was performed as described by Fredriksson et al. (16) with some modifications for the handling of small samples as described by Frayn et al. (17). Sufficient material could be obtained for the assay in both adipose regions from 12 subjects. Packed fat cells (exactly 300 µL) that had been stored in liquid nitrogen were homogenized at 4 C in 2.0 mL of a buffer containing 0.25 mol/L sucrose, 1 mmol/L ethylenediamine tetraacetate, 1 mmol/L dithiothreitol, and 20 µg/L of each of the protease inhibitors antipain and leupeptin. The homogenate was then centrifuged at 100,000 x g at 4 C for 45 min. The fat cake was removed, and the fat-free infranatant was recovered for analysis of HSL activity using 1(3)-[3H]-oleoyl-2-O-oleylglycerol as substrate (16). The substrate was obtained from the same source (Department of Medical and Physiological Chemistry, Lund University, Lund, Sweden) as in the original methodological studies. All samples were incubated in triplicate at 37 C for 30 min on one occasion. The substrate for HSL has only one hydrolyzable ester bond at the 1(3) position and is therefore not a substrate for monoacylglycerol lipase, which is abundant in adipose tissue. Furthermore, under the present incubation conditions (pH 7.0 and no apolipoprotein CII present), lipoprotein lipase activity is negligible (16). As the phosphorylated and dephosphorylated forms of the enzyme have the same activity toward this substrate, the total amount of activatable enzyme in the sample is measured (17). HSL hydrolyzes tri- and diacylglycerol at the relative rate of 1:10 (16). Therefore, the sensitivity of the assay is enhanced by the use of a diacylglycerol analog as substrate. One unit of enzyme activity equals 1 µmol fatty acid produced/min at 37 C. Enzyme activity was related to the cell number of the sample, which was determined as described above. The enzyme activity of the fat-free infranatant was proportional to the volume of packed fat cells when multiple samples from one subject were analyzed (r = 0.94; P < 0.01). The within-run coefficient of variation was 7%. The intraindividual coefficient of variation, examined by multiple samples of adipose tissue from one subject, was 11%.
Assay of mRNA levels
Steady state mRNA levels of HSL were measured using a solution hybridization assay in 19 subjects (18, 19). Packed adipocytes (200 µL) were homogenized. and total nucleic acids (TNA) were extracted by phenol-chloroform after digestion with proteinase K (18).
A 453-bp fragment corresponding to nucleotides 1865–2318 of the human HSL complementary DNA (6) was cloned into a pBluescript vector and linearized using EcoRI. Antisense RNA produced using T3 RNA polymerase was labeled by incorporating [35S]UTP into the reaction mixture (17). The specificity of the probe has been ascertained previously (17). An unlabeled sense RNA complementary to the probe was synthesized and used as control. The concentration of the RNA was determined by spectrophotometry.
The radiolabeled probe was hybridized to the samples or the sense RNA for 18 h at 68 C in a buffer containing 25% formamide (19). After hybridization, the samples were treated with ribonuclease for 45 min at 37 C and then precipitated for 30 min at 4 C after addition of trichloroacetic acid. The ribonuclease-resistant precipitated RNA-RNA hybrids were collected under vacuum on a Whatman GF/C filter (Whatman, Clifton, NJ) that was washed with 4% trichloroacetic acid in 1% sodium pyrophosphate and with 70% ethanol before scintillation counting (19).
The HSL mRNA level was related to the content of 
-actin in the
nucleic acid extract, measured with the solution hybridization
technique described above using a 236-bp probe, donated by Mats
Gåfvels, Novum, Huddinge Hospital (Huddinge, Sweden).
Drugs and chemicals
Collagenase prepared from Clostridium histolyticum and BSA (fraction V, lot 63F-0748) were obtained from Sigma Chemical Co. (St. Louis, MO). Isoprenaline was purchased from Hässle (Molndal, Sweden). Glycerol kinase from Escherichia coli (Sigma no. G4509) and ATP monitoring reagent containing firefly luciferase (LKB Vallac, Turku, Finland) were used in the glycerol assay. All other chemicals were commercially available and of the highest grade of purity.
Statistical analysis
Student’s two-tailed t test was used for comparison between adipose regions. Simple regression analysis and analysis of covariance were used in some cases. Data are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The results of the lipolysis experiments are summarized in Fig. 2. The basal lipolysis rate,
i.e. in the absence of agonist, was more than 2-fold higher
in the sc compared to the omental adipocytes. The maximum stimulatory
effect of the different lipolytic agents used was also greater in the
sc compared to the omental adipocytes, although the difference was less
pronounced than that for basal lipolytic activity. The increase in the
lipolysis rate in sc adipocytes was of the same order of magnitude
whether lipolysis was stimulated at the ß-adrenoceptor level with
isoprenaline or at postreceptor levels with forskolin or dcAMP.
Furthermore, the higher lipolytic capacity in the sc region was also
found when the basal values were subtracted and when the results are
expressed in relation to lipid weight (data not shown).
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. Enzyme activity was about 80% higher
in the sc compared to the omental samples. This increase corresponds to
the increase in stimulated lipolytic activity. HSL mRNA levels were
higher in TNA extracts from sc compared to omental adipocytes (Table 1). The difference was, however, less pronounced than that for HSL
activity. There were no significant correlations between HSL mRNA
levels, on the one hand, and HSL activity, lipolysis rate, or fat cell
size, on the other (data not shown).
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). There was a significant correlation
between fat cell size and maximum isoprenaline-stimulated lipolysis in
each adipose region; the regression lines of the two regions were
practically superimposed. A similar pattern was observed when HSL
activity was plotted against the maximum lipolysis rate (Fig. 3b).
Analysis of covariance revealed no significant difference between the
regression lines in either case. It is known that variations in fat
cell size depend not only on body mass and adipose region but also on
gender. Therefore, in Fig. 4 we have
compared the regression lines between cell size and lipolysis in males
and females for both sc and omental adipocytes taken together. There
were no significant differences between the regression lines as
determined by analysis of covariance.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Previous studies of lipolysis in man have described a stronger
lipolytic response to catecholamines in omental cells due to
differences at the adrenoceptor level with higher density and thus
sensitivity to ß-adrenoceptor stimulation in visceral fat combined
with reduced 2-adrenoceptor expression (22). Receptor
affinity for other antilipolytic agents of importance, such as
adenosine and insulin, is higher in sc than in visceral fat, further
promoting lipolysis from the visceral region (20, 22). However,
postreceptor differences and HSL expression have never been studied in
man.
A positive relationship was found between lipolytic capacity and fat cell size in the two adipose depots. This observation is in accordance with previous findings (10, 20, 23, 24). It may also explain discrepancies between the present study and previous studies in man and the rat. In severely obese subjects, no difference was found between the lipolysis rates of omental and sc fat cells (25, 26). There were also negligible differences in cell size between the two regions. In the rat, sc adipocytes are characterized by lower maximal lipolysis rate and HSL expression than internal adipocytes. In this species, sc adipocytes are smaller than internal adipocytes (8). From these data it may be speculated that under physiological conditions, the lipolytic capacity and the level of HSL are at least partly determined by fat cell size. In agreement with this hypothesis, the regression lines for the relationship between cell size and lipolysis rate were nearly superimposed for the two adipose regions. It is possible that the increased lipolytic capacity and HSL activity observed in large fat cells is an adaptive mechanism to limit fat cell hypertrophy. This could perhaps explain the decreased HSL activity in sc adipocytes of obese women subjected to a weight reduction program that induced a marked reduction in fat cell size (27). Cell size is probably not the only determinant of maximal lipolytic capacity and HSL expression. In pathological conditions such as familial combined hyperlipidemia and insulin resistance, other factors are probably involved (28, 29).
The exact mechanism behind the increase in HSL activity in the enlarged
sc adipocytes can only be speculated upon. Unfortunately, the small
amounts of adipose tissue available did not permit direct analysis of
HSL protein expression. We did, however, find increased expression of
HSL mRNA in sc adipocytes, suggesting that the regional differences at
least partly may be determined at a transcriptional level. However, the
increase in mRNA levels was less pronounced than the regional
differences in HSL activity or lipolysis. Furthermore, we found no
significant correlations between mRNA levels and HSL activity or
lipolysis rate. This may be interpreted in several ways. First, there
may be several different regulatory mechanisms involved. Second, the
use of -actin mRNA as a reference may cause an underestimation of
the HSL mRNA levels. There is evidence that the total amount of RNA is
greater in large cells than in small cells derived from different
tissues (30). Also, it appears that the expression of -actin is
correlated to the amount of total cellular RNA (31). However, to our
knowledge, this has not been examined in cells of the same origin and
degree of differentiation as is the case with adipocytes from
individuals with varying degrees of obesity. It should be kept in mind
that in adipocytes, the expanded cytoplasm is mainly occupied by the
enlarged lipid droplet, and it is uncertain to what degree the increase
in fat cell size might influence RNA expression. A measurement of the
ratio of RNA to DNA content in the TNA samples could shed some light on
these questions. Unfortunately, the amount of tissue available was too
small to allow precise determinations of DNA content. To further
examine the relationship between cell size and regulation of lipolysis
rate would require detailed studies of both transcription and
translation, which were beyond the scope of this study.
There was no selection on the basis of gender in this study. Thus, although the study included subjects of both sexes, it did not allow direct comparison between male and female subjects because these were not matched for age or body mass index. Previous studies have suggested, however, that there are gender differences regarding the regional variation in lipolysis rate and fat cell size. In women, the increase in cell size and lipolytic rate in sc as compared to omental adipocytes is maintained with increasing body weight, but appears to be abolished in obese men (24, 25, 32, 33). However, concerning the close correlation observed between cell size and lipolysis rate, we did not find any difference between men and women. This suggests that the factors determining the relationship between cell size and HSL activity are not dependent on gender.
In summary, the present study investigated the possible role of HSL in determining regional differences in lipolytic capacity. We found larger fat cells and higher lipolysis rates, HSL activity, and mRNA expression in sc compared to omental adipocytes. Variations in fat cell size appear largely to explain these differences, as lipolysis rates correlate with fat cell size regardless of the adipose region.
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Acknowledgments |
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Footnotes |
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Received January 16, 1997.
Revised July 23, 1997.
Accepted August 12, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-actin
gene: differential evolution, location and expression of the
cytoskeletal ß- and -actin genes. Mol Cell Biol. 8:1775–1789.
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