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Karolinska Institute, Clinical Research Center and Departments of Medicine (P.L., J.H., M.R., H.W., P.A.) and Surgery (A.T.), Huddinge University Hospital, S-141 86 Stockholm, Sweden; and Department of Cell and Molecular Biology, Lund University (C.H.), S-22100 Lund, Sweden
Address all correspondence and requests for reprints to: Peter Arner, M.D., Karolinska Institute, CME M61, MK Division, Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail: peter.arner{at}medhs.ki.se
Abstract
The influence of obesity on the lipolytic capacity of isolated sc fat cells was studied prospectively in 13 women and 10 men, all obese, but otherwise healthy, before and 2 and 3 yr after weight reduction by bariatric surgery. Nonobese subjects (25 women and 17 men) without a family history of obesity served as the control group. Lipolytic capacity was determined after stimulation at different steps of the lipolytic cascade with noradrenaline, isoprenaline, forskolin, and (Bu)2AMP. Bariatric surgery was followed by a marked and similar reduction of body mass index and fat cell volume (
40%) in both genders. Before weight loss, lipolytic capacity per cell was elevated in obese women and decreased to normal levels after weight reduction at 2 and 3 yr. However, lipolytic capacity per fat cell surface area was not changed in obese women. In obese men, lipolytic capacity per cell was almost the same as in lean men and was not influenced by weight reduction. Lipolytic capacity was related to fat cell size in women (P = 0.0008; r = 0.58), but not in men (P = 0.67; r = 0.086). The protein content of hormone-sensitive lipase, which determines lipolytic capacity, was significantly lower in obese men and women and increased slightly after weight reduction in men only. Thus, in women, but not in men, the adipocyte lipolytic capacity is influenced by obesity and weight reduction, probably due to changes in fat cell size. These gender differences are not related to the amount of hormone-sensitive lipase protein in adipocytes.
HUMANS SHOW CHARACTERISTIC sex-specific differences in the development and distribution of adipose tissue to different regions of the body. In obese men excess adipose tissue is located primarily in the abdominal region. Women, on the other hand, tend to also accumulate excess adipose tissue in the gluteo-femoral regions (1). Numerous studies have emphasized the idea that regional adipose tissue distribution, particularly abdominal fat deposition, is strongly correlated to the metabolic complications of obesity, such as hypertension, cardiovascular disease, and noninsulin-dependent diabetes mellitus (2, 3, 4, 5). Thus, it appears that men are at higher risk of developing complications related to obesity than are women.
Lipolysis (the hydrolysis of triglyceride into FFA and glycerol) in humans is above all stimulated by catecholamines. The catecholamine effects are modulated through four subtypes of adrenoceptors, i.e. stimulation via ß1-, ß2-, and ß3-adrenoceptors and inhibition via 
2-adrenoceptors (6). Thus, the final response of adipose tissue to catecholamines depends on the functional balance between inhibitory and stimulatory receptors (7). Catecholamine receptors regulate cAMP formation. This modulates activation of hormone-sensitive lipase (HSL), which is the final rate-limiting step of lipolysis. The amount of HSL appears to be an important determinant of the lipolytic capacity in human sc fat cells (8).
As previously reviewed (6), a number of abnormalities of catecholamine-induced sc adipose lipolysis are observed in obesity. Although the spontaneous, basal rate is increased, the lipolytic effect of catecholamines is decreased. The latter seems to be due to at least three defects, namely increased 2-antilipolytic function, decreased ß2-adrenoceptor lipolytic function, and a decreased ability of cAMP to activate lipolysis (i.e. decreased lipolytic capacity). Another important factor for adipocyte lipolysis is fat cell size, which correlates with the lipolytic rate, and where obese subjects have much larger fat cells than lean individuals (9). When lipolysis rates are expressed per cell, the values for maximal stimulation are higher in obese than in nonobese subjects (10). However, when the effect of increased cell size is taken into account, the lipolytic capacity in obese fat cells is lower than expected for the fat cell size (10).
Some important issues on sc fat cell lipolysis in obesity remain to be determined. For example, is the influence of obesity on lipolytic capacity different between men and women, and which abnormalities of lipolysis are primary and secondary? To the best of our knowledge the gender aspect has not previously been investigated. The question about primary defects in lipolysis regulation can be answered from studies of weight reduction. Only short-term and moderate weight reduction has been studied to date (11, 12). It is unclear whether these effects relate to weight reduction per se or to the hypocaloric diet (13, 14, 15, 16).
The present study was undertaken to elucidate the effect of obesity and the influence of long-standing major weight reduction on the ability to maximally activate lipolysis (i.e. lipolytic capacity) in abdominal sc fat cells of obese men and women. Although lipolysis regulation may differ between peripheral and central sc regions (16, 17), we chose the abdominal site because of its strong relation to metabolic aberrations in obesity (2, 3, 4, 5). Patients were studied before and at 2 and 3 yr after weight reduction surgery using the adjustable gastric banding technique (18). The maximum lipolytic effect of the natural catecholamine noradrenaline and of drugs that can activate lipolysis at different steps of the lipolytic cascade were studied and set in relation to the adipocyte content of HSL.
Subjects and Methods
Patients
The study group consisted of 23 obese but otherwise healthy subjects scheduled for weight reductive surgery at Huddinge University Hospital (10 men and 13 women). Ages ranged from 24–57 yr, and body mass index (BMI) ranged from 30–52 kg/m2. There were no significant differences between the two genders in age or BMI (see Table 1
for additional clinical data).
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None of the patients or controls was taking regular medication. All were Scandinavians. All subjects had given their informed consent before entering the study. The study was approved by the ethics committee of Karolinska Institute.
Experimental procedure
A prospective study design was used. The study group consisted of patients accepted for surgical treatment of obesity using the adjustable gastric banding technique (18). They were examined before and at regular intervals after surgery for routine clinical measurements, including BMI and waist/hip ratio (W/H). All 23 subjects completed the first 2 yr of the study. Before and 2 yr after operation they were examined at the research laboratory according to the experimental protocol. For 10 patients it was possible to perform an additional examination 3 yr after surgery. The other 13 subjects left the study for reasons of compliance or did not show up at the scheduled 3-yr visit. The control group was examined in exactly the same way as the study group, but on only one occasion.
Subjects were examined in the morning after an overnight fast. Height, body weight and W/H were measured. After a 15-min rest in the supine position, venous blood samples were drawn for the subsequent analysis of plasma glucose at the routine chemistry laboratory of the hospital. Plasma insulin was also measured using a commercial RIA kit (Amersham Pharmacia Biotech, Uppsala, Sweden).
Thereafter, sc adipose tissue (1–2 g) was obtained from the paraumbilical region by needle aspiration under local anesthesia using 5–10 ml 0.5% lidocaine as previously described (20). It has previously been demonstrated that this procedure does not influence adipocyte metabolism (20). The tissue samples were immediately transported to the laboratory. One piece (300 mg) was rinsed in saline, immediately frozen in liquid nitrogen, and stored at -70 C for subsequent protein analysis.
None of the obese subjects reported important weight changes during the 4 wk proceeding each fat biopsy.
Isolation of fat cells and lipolysis experiments
Isolated fat cells were prepared and isolated according to Rodbell (21). In brief, adipocytes were separated from stromal cells by treatment in a shaking bath at 37 C for 60 min with 0.5 mg/liter collagenase in 5 ml Krebs-Ringer phosphate buffer (pH 7.4) with 40 g/liter purified BSA. Adipocyte suspensions were then rinsed three times in collagenase-free buffer using nylon filters. Fat cell sizes were measured by direct microscopy, and the mean adipocyte diameter was calculated from measurements of 100 cells. Because adipocytes have 95% lipid content and are spherical in shape, volume and weight can be estimated from the diameter (22). The total lipid weight of the incubated fat cells was determined after organic extraction. The number of fat cells incubated was determined by dividing total lipid weight by fat cell weight.
The lipolysis assay has previously been described in detail (23). In brief, a diluted suspension of isolated fat cells (5–10000 cells/ml) was incubated for 2 h in duplicate samples with air as the gas phase at 37 C in Krebs-Ringer phosphate buffer (pH 7.4) supplemented with glucose (1 mg/ml), ascorbic acid (0.1 mg/ml), and BSA (20 mg/ml) in the absence (basal) or presence of increasing concentration of different stimulators of lipolysis. The latter included noradrenaline, which is a natural nonselective - and ß-agonist; isoprenaline, a nonselective ß-adrenoceptor agonist; forskolin, which stimulates adenylyl cyclase; and (Bu)2cAMP, which is a phosphodiesterase-resistant cAMP analog that stimulates the PKA-HSL complex.
After the incubation, an aliquot of the medium was removed, and glycerol, which was used as a measurement of the lipolysis rate, was analyzed using a bioluminescence method (24). All agents caused a concentration-dependent stimulation of lipolysis that reached a plateau at the highest concentrations of agonist in each individual experiment. The rate of lipolysis was expressed as micromoles of glycerol per 107 cells/2 h and as micromoles of glycerol release per cell surface area. The formula used to calculate cell surface area has been discussed in detail previously (10, 25). We calculated the rate of glycerol release at maximum effective concentration for each of the lipolytic agents used (lipolytic capacity).
Protein isolation and Western blot analysis
The amount of HSL protein in adipose tissue was determined as described previously (10). Frozen sc adipose tissue (300 mg) was crushed, lysed in protein lysis buffer [1% Triton X-100, Tris-HCl (pH 7.6), and 150 mM NaCl] supplemented with protease inhibitors [1 mmol/liter phenylmethylsulfonylfluoride and Complete (Roche Molecular Biochemicals, Mannheim, Germany)], and homogenized. The homogenate was centrifuged at 14,000 rpm for 30 min, and the infranatant was removed and saved. All steps were performed at 4 C to minimize the risk of protein degradation. The protein content in each sample was determined using a kit of reagents from Pierce Chemical Co. (Rockford, IL). One hundred micrograms of total protein were then loaded on polyacrylamide gels and separated by standard SDS-PAGE. To control for differences in gel migration, exposure time, antibody incubation, etc., samples from obese and control subjects were run on the same gels and transferred to the same polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). Blots were blocked for 1 h in room temperature in Tris-buffered saline with 0.1% Tween 20 and 5% nonfat dried milk. This was followed by an overnight incubation at 4 C in the presence of antibodies directed against HSL. HSL antibodies were generated by one of the authors (C.H.). Secondary antibodies conjugated to horseradish peroxidase were obtained from Sigma (St. Louis, MO; -rabbit, 1:4000; -chicken, 1:2500). Antigen-antibody complexes were detected by chemiluminescence using a kit of reagents from Pierce Chemical Co. (Supersignal), and blots were exposed to high performance chemiluminescence film (Amersham Pharmacia Biotech). Films were scanned, and the OD of each specific band was analyzed using the program Image (NIH, Bethesda, MD) and expressed as OD per mm2/100 µg total protein.
Drugs and chemicals
BSA (fraction V, lot 63F-0748), Clostridium histolyticum collagenase type I, forskolin, (Bu)2cAMP, and glycerol kinase from Escherichia coli (G4509) were obtained from Sigma. Isoprenaline was obtained from Hässle (Molndal, Sweden). ATP monitoring reagent containing firefly luciferase was purchased from LKBWallac, Inc. (Turku, Finland). All other chemicals were of the highest grade of purity commercially available.
Statistical methods
Data are presented as the mean ± SEM and were compared using paired or unpaired t test and ANOVA. Calculations were made using the StatView software program (Abacus Concepts, Berkeley, CA).
Results
Clinical data
The characteristics of the study groups are presented in Table 1
. At baseline obese men and women showed classical abnormalities. BMI at baseline was increased in women (45 ± 1) compared with men (40 ± 1; P = 0.02). W/H was significantly lower in women (0.92 ± 0.02) than in men (1.04 ± 0.04; P = 0.0002), whereas fat cell volume and fasting plasma insulin and glucose did not differ between genders at baseline (P values from 0.26–0.86). All of these parameters improved markedly 2 yr after bariatric surgery. BMI, being somewhat higher at baseline in females decreased to about the same level for both genders after bariatric surgery at 2 yr (men, 33 kg/m2; women, 32 kg/m2; P = 0.7). There was no significant difference in fat cell volume, plasma glucose, or plasma insulin after weight loss between genders (P = 0.86, P = 0.57, and P = 0.41, respectively). The range of BMI loss after surgery was 3.5–25.0 kg/m2 at 2 yr and 7.2–25.0 kg/m2 at 3 yr in women and 3.3–19.0 kg/m2 at 2 yr and 6.9–25.0 kg/m2 at 3 yr in men.
Lipolysis
Figure 1 shows for illustrative purposes the mean concentration-response curves for noradrenaline (lipolysis per cell) in men and women before and 2 yr after weight loss compared with lean controls. In all experiments noradrenaline increased lipolysis in a concentration-dependent fashion. In women the mean baseline curve of the obese subjects was elevated compared to that in lean subjects and was also normalized after weight loss. However, in men the three mean curves did not differ much in their positions in the graph.
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Group data for lipolysis were also expressed per cell surface area (Table 3). Obese men showed no difference from lean men in lipolysis either before or after weight reduction, although stimulated lipolysis actually improved (at a borderline significant level) after the decrease in body weight. In obese women basal lipolysis was increased at baseline and was completely normalized after weight reduction. Stimulated lipolysis was similar in obese and nonobese women, although it decreased (at a borderline level of significance) after surgery.
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. BMI decreased gradually in men and women, with the major part of the decrease occurring from 0–2 yr. In women the rate of basal and (Bu)2cAMP-induced lipolysis decreased markedly from 0–2 yr and was thereafter almost constant. However, in men the lipolytic rates were constant from 0–3 yr. Similar results were obtained with isoprenaline and forskolin (values not shown).
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for lipolysis values). There was no significant difference in stimulated lipolysis between genders after weight loss (P values ranging from 0.39–0.72). However, basal lipolysis was higher in men than in women after weight loss (P = 0.05). There were no important gender differences in lipolysis in nonobese subjects.
The relationship between fat cell size and lipolytic capacity was examined using simple regression analysis for obese and lean patients (Fig. 3). There were significant correlations between fat cell size and basal lipolysis both in men and women (Fig. 3, A and B). In Fig. 3B four obese men showed very low lipolysis. However, these outliers responded to weight loss in the same way as the other men, and when they were omitted in statistical analysis, the results were the same as when they were included. However, a gender difference was observed when studying maximally stimulated lipolysis. The maximum sc lipolysis induced by (Bu)2cAMP, forskolin, or isoprenaline showed a strong correlation to fat cell size in women, but there was no correlation in men. Fig. 3, C and D, shows the data with (Bu)2cAMP. Similar results were obtained with isoprenaline and forskolin (graphs not shown). In women, the reduced basal and stimulated lipolysis values after weight reduction were distributed evenly along the same regression line as the baseline values.
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To evaluate the expression of proteins involved in lipolytic activity after weight reduction, we obtained sc adipose tissue samples from a limited set of subjects (7 men and 10 women) before and after weight reduction. The samples were run on the same Western blot (1 for men and 1 for women). To evaluate absolute amounts of detected protein we also compared sc adipose tissue from lean control subjects (8 men and 8 women) with the obese samples obtained before weight reduction. The control subjects were chosen at random. Obese and lean men were run on one Western blot, and female samples on another. Total cytosolic protein was isolated and separated by Western blotting. Immunodetection was performed with antibodies directed against HSL.
The adipose HSL content was significantly reduced by about 50% in obese compared with lean subjects (P = 0.003 for men and P = 0.001 for women; OD per mm2/100 µg total protein ± SE: obese men, 555 ± 95; lean men, 1401 ± 140; obese women, 422 ± 57; lean women, 942 ± 179). With regard to the effect of weight reduction at 2 yr the amount of HSL protein (OD per mm2/100 µg total protein ± SE) was slightly increased in men (925 ± 120 before and 1169 ± 93 after; P = 0,045), but not in women (695 ± 120 before and 892 ± 149 after; P = 0.1).
Discussion
In the present study marked differences in the influence of obesity and subsequent body weight reduction on lipolysis regulation were seen between women and men. In obese women the basal rate of lipolysis as well as the lipolytic capacity (measured by agents acting at various levels of the lipolytic cascade) were markedly increased when expressed per fat cell. However, lipolytic capacity per cell surface area was similar in obese and nonobese women. A marked weight reduction (to a level still considered as obese, 32 kg/m2) completely normalized basal lipolysis, lipolytic capacity, and noradrenaline-induced lipolysis (expressed per cell) in women. It is well known that the lipolytic rate in fat cells is related to fat cell size. It is very likely that the influence of obesity and weight reduction in women above all is related to changes in fat cell size, which is increased in obesity and normalized after weight reduction. Firstly, in the whole group of women there was a strong relationship between lipolysis rate per cell and fat cell size. Secondly the rates of lipolysis per cell in adipocytes of weight-reduced obese women were distributed along the same regression line as baseline values.
In obese men the basal rate of lipolysis per cell was increased, but, in contrast to women, it was not influenced by weight reduction despite the fact that fat cell size decreased after bariatric surgery to the same extent in men as in women. The lipolytic capacity and the effect of noradrenaline on lipolysis were not influenced by either obesity or weight reduction when expressed per cell. Furthermore, there was no relationship between lipolytic capacity per cell and fat cell size in men. Lipolysis per cell surface area was not influenced by obesity in men, although it tended to improve after weight reduction.
These data strongly indicate that obesity influences lipolysis in the sc abdominal site in a different manner in men and women. The increased rate per cell, but not per cell surface area, observed in women indicates that obesity-mediated changes in women probably are due to the change in fat cell size. In men the increase in the basal rate of lipolysis might be a primary defect (or at least more resistant to weight reduction). It is possible that the gender difference in lipolytic capacity could be due to the fact that women adapt their lipolysis to obesity. The increase in lipolytic capacity in obese women could be a protective phenomenon. Men do not seem to be able to increase their lipolytic capacity in sc abdominal adipose tissue when becoming obese. This might at least in part explain why men are more prone to accumulate fat in the abdominal region than are women. The observation that lipolysis per cell differed between obese men and women, but not between lean subjects of either sex, further suggests that obesity influences lipolysis differently in males and females.
A lower maximal lipolytic capacity in sc abdominal adipocytes in obese vs. lean subjects has previously been reported (10). The previous study (10), in contrast to the present, was conducted on a mixed population of both men and women. Furthermore, the controls in the present study [as opposed to the previous one (10)] had no family history of obesity. It has been shown that family history of obesity influences lipolysis in lean subjects (26).
Data from basal lipolysis were somewhat different from those from stimulated lipolysis. It should be noted that the meaning of basal lipolysis measured in vitro is unclear, as fat cells in vivo always are exposed to regulatory hormones. However, the fact that basal lipolysis in obese women was elevated when expressed per cell and per cell surface area may suggest that it is not influenced by fat cell size.
It should be stressed that our findings may only relate to sc abdominal fat. Important regional differences in lipolysis within sc adipose tissue and between this region and the visceral one have been reported (6, 7). For ethical reasons it is not possible to investigate visceral fat in this type of study.
In previous in vitro studies of lipolysis regulation after weight reduction, obese patients have usually been investigated after short-term and moderate weight loss has been achieved. Conflicting results showing unchanged (12, 14, 15), enhanced (13, 16, 25, 28), or reduced (29) catecholamine-induced lipolysis (12, 13, 14, 15, 16, 25, 28, 29) after weight loss have been reported. The differences in results between previous studies might be explained by moderate weight loss (10 kg on the average) and that the duration of experiments has been limited to 10–15 wk. Some effects may also be related to the diet itself rather than to weight loss. In the present study we investigated the effect of a marked (32 kg) long-term weight loss. Our findings were similar when subjects were investigated 2 yr after bariatric surgery, when there was a dramatic weight reduction, and 1 yr thereafter, when the additional weight loss was very small. This indicates that the findings were not secondary to decreased caloric intake, but, rather, to weight loss per se.
In an attempt to find the mechanisms responsible for the lipolytic capacity results we examined the protein content of HSL. As similar results were obtained with isoprenaline (acting on ß-adrenoceptors), forskolin (acting on adenylyl cyclase), and (Bu)2cAMP (acting on PKA), it is likely that our findings relate to events at or near HSL. In both obese men and women, the adipose amount of HSL was markedly decreased, confirming data on mixed obese populations (10). Furthermore, the amount of HSL protein was influenced by weight reduction. In men it was slightly decreased. In women, however, no significant change was observed. It is clear that when HSL and lipolysis data are combined it is not possible to explain the gender variation on the basis of total amount of HSL protein. There are, though, a number of other possible explanations for the gender difference related to HSL, such as phosphorylation status and translocation of the enzyme. Unfortunately, other mechanistical events could not be explained because of the lack of tissue. Furthermore, the roles of as yet unidentified lipases other than HSL (30) remain to be established.
In conclusion, in obesity the lipolytic capacity of abdominal sc fat cells is increased in women due to secondary factors, but is not changed in men. This might be of importance for the difference in body fat distribution between obese men and women.
Acknowledgments
The skillful technical assistance of Britt-Marie Leijonhufvud, Catharina Hertel, Eva Sjölin, and Kerstin Wåhlén is appreciated.
Footnotes
This work was supported by grants from the Swedish Medical Research Council, the Swedish Diabetes Association, the Juvenile Diabetes Foundation, the Wallenberg Foundation, the Swedish Heart and Lung Foundation, Karolinska Institute, King Gustav V and Queen Victoria Foundation, the Belvén Foundation, the Bergvall Foundation, the Novo Nordisk Foundation, the AMF Research Fund, the Swedish Society of Medicine, and the Foundation of Thuring.
Abbreviations: BMI, Body mass index; HSL, hormone-sensitive lipase; W/H, waist/hip ratio.
Received May 31, 2001.
Accepted October 26, 2001.
References
-adrenergic responsiveness of obese human subcutaneous adipocytes. Int J Obesity 5:145–153[Medline]
2-adrenoceptors and adrenergic adipose tissue responsiveness after moderate hypocaloric diet in obese subjects. Int J Obesity 13:99–110[Medline]
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), and female controls (• on dotted line). Bottom, Obese men before weight loss (
) and after weight loss (
) and male controls (
, lean controls.