¹Department of Clinical Nutrition, University of Kuopio and Kuopio University Hospital, Kuopio, Finland

²INSERM U449, Faculté de Médecine R Laennec, Lyon, France

³Institute of Biomedicine, University of Helsinki, Finland

⁴Department of Surgery, Kuopio University Hospital, Finland

Correspondence to: M Kolehmainen, University of Kuopio, Department of Clinical Nutrition, PO Box 1627, FIN-70211 Kuopio, Finland. E-mail: Marjukka.Kolehmainen@uku.fi

OBJECTIVE: The effect of weight reduction on hormone sensitive lipase (HSL) and lipoprotein lipase (LPL) gene expression and their relationship with adipose tissue metabolism were studied in massively obese men and women.

SUBJECTS: Seventeen obese subjects (eight men, nine women) participated in the study (age 44±2 y, weight 145±8 kg, fat 40±2% of body mass, mean±s.e.m.), who were going through a gastric-banding operation for weight reduction.

MEASUREMENTS: HSL and LPL mRNA expressions were analyzed using the reverse transcription competitive polymerase chain reaction. Subcutaneous fat lipolysis was measured in vivo by microdialysis and in vitro in isolated subcutaneous abdominal adipocytes. Measurements were done before and after 1 y of weight reduction.

RESULTS: Significant reductions in weight (for men -20.3±2.5%, for women -18.3±2.1% (mean±s.e.m.) and fat mass (for men -27.6±7.9%, for women -21.8±3.9%) were observed in both genders. In women HSL mRNA expression decreased by 31% (P=0.008) and LPL expression increased slightly, but nonsignificantly (42%, P=0.110). These changes were not observed in men. In men, inhibition of lipolysis with ₂-adrenergic and adenosine agonist was improved (P=0.001) in isolated adipocytes.

CONCLUSIONS: This study uncovers new differences between genders in adipocyte metabolism along with weight reduction. In women, the observed changes in HSL and LPL gene expression suggest that deposition of lipids into adipose tissue might be favored after weight reduction. In men, the results indicate improved responsiveness to inhibition in adipose tissue metabolism along with weight reduction.

International Journal of Obesity (2002) 26, 6-16. DOI: 10.1038/sj/ijo/0801858

weight reduction; hormone sensitive lipase; lipoprotein lipase; lipolysis; gastric banding

Introduction

Lipoprotein lipase (LPL) and hormone sensitive lipase (HSL) are enzymes that regulate deposition and mobilization of fatty acids in adipose tissue in a reciprocal manner.¹ LPL hydrolyses circulating triglycerides from which free fatty acids are taken by adipocytes and reesterified for storage, whereas HSL is the rate-limiting enzyme of the lipolysis cascade leading to release of free fatty acids and glycerol from adipocytes. The central role of LPL and HSL in lipid deposition and mobilization has lead to speculations that defects in their expression or activity could be associated with susceptibility, development or maintenance of obese state.^2,3,4,5

In obesity, a higher activity of LPL has been found compared to normal weight subjects in some studies,^6,7 while others have shown a blunted LPL activation in obese insulin resistant subjects.^8,9 In addition, conflicting data exist when LPL activity has been measured after weight reduction. Some studies have shown an increase,^10,11 some no change or even a decrease¹² in LPL expression and activity after weight loss. Regarding HSL, its activity and expression have also been found altered in obesity. Several studies have shown that the lipolytic effect of catecholamines is attenuated in obese subjects,^13,14,15 which may be due to a defect in the lipolytic cascade that normally leads to the activation of HSL. Decreased HSL expression and activity have been observed in obese subjects⁵ and in the first-degree relatives of obese subjects.¹⁶ However, the activity of HSL was found to be increased in other studies involving subjects with established obesity,^17,18 a result that is in line with increased spontaneous lipolysis in subcutaneous adipose tissue of obese subjects.^14,19,20,21 As for LPL, studies on the effect of weight reduction on HSL have yielded conflicting results, with either decrease, increase or, in some studies, no change in activity or expression levels of HSL.^22,23,24

This discrepancy in response of LPL or HSL activity and expression to weight change may partly be due to the fact that data on men and women have either been analyzed together or genders have been studied in separate studies. In addition, there are only a few studies that have shown gender difference in LPL activity and expression^25,26 in obesity or have compared LPL expression between genders along with weight reduction.¹² Gender differences have been observed in lipolytic responses as well as in fatty acid uptake of adipose tissue,^{27,28,29,30,31} although the difference in metabolic changes between genders due to weight reduction does not seem to be so evident.^32,33

All these data suggest that the relative contribution of LPL and HSL to the development of the fat mass and its reduction during weight loss is still controversial and largely unclear. To the best of our knowledge, there are no studies in which weight loss-induced changes in HSL and LPL expression and in the metabolism of adipose tissue have been studied in parallel in the same obese subjects. The purpose of the present work was to investigate adipose tissue metabolism before and after weight reduction in the same obese subjects to clarify the possible relationship between the RNA expression level of HSL and LPL and metabolic data from cellular and tissue level. Changes in adipose tissue metabolism were studied in vivo by a microdialysis and in vitro in isolated adipocytes, and changes in HSL and LPL mRNA levels in adipose tissue biopsies were studied by reverse transcription reaction followed by competitive polymerase chain reaction (RT-cPCR).

Methods

Subjects

Seventeen massively obese subjects (eight men and nine women) participated in the study. The subjects were going through medical examinations before a gastric banding operation for weight reduction. All the subjects were normoglycemic based on an oral 2 h glucose tolerance test (OGTT) with a glucose load of 75 g performed 7-56 days before the gastric banding operation. Two of the subjects had a cardioselective -blocking medication (atenolol). All subjects received both oral and written information on the study and they signed a written consent. The Ethics Committee of the Kuopio University Hospital and the University of Kuopio approved the study.

Study design

The subjects visited the out-patient clinic of the Kuopio University Hospital before the gastric banding operation (84-168 days before) and 1 y after it. At both visits, body composition was determined by a bioelectrical impedance method (Body Comp II, Version 1.5, RJL Systems Inc., Detroit, MI, USA) in fasting condition. Two microdialysis studies were carried out 1 week before and 1 y after the operation in the Department of Clinical Nutrition, University of Kuopio. No drug treatment was allowed for at least 24 h before the microdialysis study. Adipose tissue samples for the preparation of isolated adipocytes and for the determination of HSL and LPL mRNA levels were taken during the gastric banding operation from subcutaneous abdominal adipose tissue under general anesthesia. One year after surgery, another subcutaneous abdominal adipose tissue biopsy was taken under local anesthesia (lidocain 10%) at the out-patient clinic. Local or general anesthesia has been found not to affect adipose tissue metabolism in isolated adipocytes.³⁴ The samples for isolated adipocyte preparation were immediately placed in 0.9% saline and the isolation procedure was started within the following 30 min. Adipose tissue samples for the mRNA expression studies were immediately frozen in liquid nitrogen and stored in -70°C for later analyses.

Microdialysis

Subcutaneous adipose tissue metabolism was studied after an overnight fast, at rest, using a microdialysis method^35,36 described in detail previously.³⁷ Three microdialysis probes were inserted without local anesthesia into subcutaneous abdominal adipose tissue left from the umbilicus with the help of plastic canula (VenflonÒ2). The distance between the probes was 3-5 cm. Sterile preparations of the agents diluted into Ringer's solution were continuously perfused into the tissue (1.5 µl/min). The perfusates were collected as 20 min fractions.

The microdialysis study lasted for 3½ h. During the first hour, Ringer's solution was perfused into the adipose tissue to monitor the basal state using the three probes. The basal state was determined from the dialysates collected during the last 40 min of the first hour in every probe separately. For studying the stimulation of lipolysis, 10 and 30 µmol/l IsoprenalineÒ was infused. Inhibition of lipolysis was studied using 1, 5 and 10 µmol/l of adenosine (AdenocorÒ) and 10 µmol/l of ₂-adrenergic agonist, clonidine (CatapresanÒ). Between each solution and different concentrations of the agent, Ringer's solution was perfused for 20 min into tissue for washout. Glycerol concentrations were measured in each fraction of the dialysate to estimate lipolysis.

Ethanol was used (250 µl of a 96% solution into 100 ml of Ringer) for the estimation of local blood flow.^36,38 Ethanol concentrations were measured both in the perfusate and the dialysate. The blood flow was estimated as ethanol clearance by dividing dialysate concentration by perfusate concentration. The level of stimulation is expressed as a ratio by dividing the isoprenaline simulated glycerol concentration in dialysate by basal glycerol concentration (measured during the last 40 min of the first hour of the study).

Biochemical measurements

Fasting blood samples for the analyses of serum glycerol, glucose, free fatty acid and leptin concentrations were obtained before and after each microdialysis study. In addition, a blood sample for measurement of fasting plasma insulin concentration was taken before the OGTT. Serum glucose was analyzed by a glucose dehydrogenase method (CV 2.5%; Merck, catalog no. 12193). Serum free fatty acids were measured by a turbidometric analyzer (CV 1.5%; Kone Ltd, Espoo, Finland). Radioimmunoassays were used for the analysis of plasma insulin (CV 6%; Phasedeph insulin RIA 100; Pharmacia Diagnostics, Uppsala, Sweden) as well as for the analysis of serum leptin (CV 13%; Linco Research Inc., St. Louis, MO, USA). Serum and microdialysis perfusate concentrations of glycerol were analyzed by a modification of the enzyme assay method of Kather and Wieland³⁹ and the NADH generated was measured fluorometrically (CV 6%). Ethanol was analyzed by an enzymatic method (CV 4%; Boehringer Mannheim, Germany, catalog no. 176 290) and conventional fluorometry.

Study of lipolysis in isolated adipocytes

Adipocytes were isolated according to the modification of Ohisalo et al⁴⁰ of the method of Rodbell⁴¹ in the presence of collagenase (0.5 mg/ml) under constant shaking at 37°C in a buffer containing 125 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl₂, 2.5 mmol/l MgCl₂, 1 mmol/l KH₂PO₄, 4 mmol/l glucose, 2% BSA and 25 mmol/l Tris at pH 7.4. After 60 min cells were filtered through nylon cloth and washed three times with the same buffer without collagenase. Median cell diameter was estimated by direct microscopy of isolated cells. In one woman the cell size could not be measured due to technical reasons. For the studies of lipolysis isolated adipocytes were diluted at 1:6 ratio into the same buffer without collagenase. 250 µl of this cell suspension, containing 40 µl isolated adipocytes, was incubated for 50 min at 37°C under constant shaking (80 strokes per min). Adenosine deaminase (1 µg/ml) was added into the incubation medium. The release of adenosine from adipocytes into the medium can sometimes be enough to cause full inhibition of lipolysis.⁴² Therefore, we added adenosine deaminase to the medium to remove endogenous adenosine and then added different concentrations of the effectors mentioned below.⁴⁰ Basal and maximally stimulated (1 mmol/l forskolin) lipolysis was determined. Different concentrations of isoprenaline, adrenaline, ₂-adrenergic agonist UK-14304 (UK) and N⁶-phenyl-isopropyladenosine (PIA) were added, as shown in the results. Boiling the samples for 2 min terminated the incubation. Glycerol release values are given as pmol/µl of cells/ min and for basal and maximally stimulated lipolysis also for cell number in the incubation, as µmol/10⁷ cells/50 min. Effective agonist concentration causing 50% of maximal effect as an apparent measure of sensitivity (EC50) values^32,43 was calculated separately for the group of women and for the group of men. Individual values could not be calculated due to large individual variation in response to different concentration of different agents used and low number of the concentrations used in the lipolysis study.

Preparation of total RNA and quantitation of target mRNAs

For total RNA preparation, adipose tissue samples were pulverized in liquid nitrogen. Total RNA from the frozen powder was prepared using RNeasy total RNA kit (Qiagen). The amount of total RNA was quantified spectrophotometrically at 260 nm. The ratio of absorption (260/280 nm) of all preparations was between 1.8 and 2.0. Total RNA was suspended into water and stored at -80°C.

The levels of HSL and LPL mRNAs were quantified by reverse transcription reaction followed by competitive polymerase chain reaction (RT-cPCR). A detailed description of the method has been published previously.⁴⁴ The construction of the competitor molecule, the validation of the assay and the sequences of the primers have been reported.⁴⁵ To improve the analysis of the competitive PCR products, the sense primers were 5^' end labeled with the CY-5 fluorescent probe.

For each mRNA assay, a specific first-strand cDNA synthesis was synthesized from 0.1 µg of total RNA with 2.5 U of thermostable reverse transcriptase (Tth DNA polymerase; Promega Corp., Charbonnier, France) and with a specific antisense primer in a final volume of 20 µl. The medium was overlaid with mineral oil and incubated in the thermocycler (Minicycler PTC150, MJ Research, Waterton, MA, USA) for 3 min at 60°C, followed by 15 min at 70°C and 5 min at 99°C as previously recommended.⁴⁴ After chilling in ice, 4 µl of water was added to the RT medium from which 20 µl were sampled for cDNA quantification by competitive PCR. The 20 µl of RT medium were added to a 100 µl PCR mix (final volume) containing 45 pmol of the corresponding sense primer and 30 pmol of the antisense primer. Four aliquots (20 µl) were then transferred to four microtubes containing 5µl of a defined working solution of the competitor cDNA. The medium was overlaid with mineral oil and PCR amplification was processed for 40 cycles in conditions that have been described in detail previously.^44,45 The PCR products were analyzed in denaturating 4% acrylamide-gel (Ready-mix, Pharmacia, Upsala, Sweden). The electrophoresis was run with an ALF Express DNA sequencer (Pharmacia). The fluoresence of the target and competitor cDNA pics was evaluated using the Fragment Manager software (Pharmacia). The logarithm of the competitor on target amount ratio was plotted vs the logarithm of the initial amount of the competitor added in the PCR. The target mRNA concentration was calculated at the competition equivalence point.⁴⁴

Statistics

All calculations were performed using the SPSS/WIN program version 8.0 (SPSS Inc., Chicago, IL, USA). Results are given as means and standard errors of mean (s.e.m.). Nonparametric statistics were used due to the small number of subjects: when analyzing biochemical and anthropometric data, the Wilcoxon test was used for studying differences before and after weight reduction within gender group and the Mann-Whitney test for studying difference between the genders. When analysing the data of adipose tissue metabolism in vivo or in vitro, a general linear model for repeated measures was used for studying the differences between genders. Analysis was corrected using cell size as a covariate. Friedman nonparametric analysis of variance test was used for testing the difference in responses before and after weight reduction within the gender. Spearman nonparametric correlation was used to study connection between certain variables of interest. When appropriate, partial correlation was used for correcting analysis with cell size and/or fat mass.

Results

Characteristics of the subjects

The anthropometric data before and after 1 y weight reduction and the changes (%) observed are shown in Table 1. Both men and women were markedly obese before the gastric banding operation. They remained obese, however, even after marked weight loss. There were no significant gender differences in the relative changes in weight, fat mass or lean body mass.

Subcutaneous cell size decreased in both genders as expected due to weight loss and reduced fat mass (Table 1). Women had larger cell size than men both before and after the operation.

The subjects had normal concentrations of serum glycerol and free fatty acids before and after the operation (Table 2). Fasting serum glucose was slightly higher before than 1 y after the operation. In women, the concentration decreased to normal limits (P=0.015), but in men there was no significant change after weight loss. Fasting serum insulin concentration decreased slightly both in men (P=0.043) and in women (P=0.015).

HSL and LPL mRNA levels before and after weight loss

The HSL mRNA level was significantly higher (P=0.018) in women than in men before the operation (Figure 1), and this difference remained significant even after correction with cell size (P=0.022). There was a marked decrease in HSL mRNA level in women after 1 y of weight reduction (323±29 vs 220±21 amol/µg of total RNA, before vs after, P=0.008). In men, no significant change in the level of HSL mRNA was found (218±27 vs 208±26 amol/µg of total RNA, before vs after, P=1.000).

Regarding LPL expression, there was no difference in LPL mRNA levels between genders in abdominal subcutaneous adipose tissue before the operation (Figure 1). Further, the change in LPL mRNA levels was not statistically significant after 1 y of weight loss in women (628±75 vs 802±121 amol/µg of total RNA, before vs after, P=0.110). In men, LPL mRNA levels did not change, either (606±63 vs 535±50 amol/µg of total RNA, before vs after, P=0.161). However, a statistically borderline difference was shown in LPL mRNA level between genders after 1 y of weight loss (P=0.054).

When analyzing the LPL/HSL mRNA ratio, a noticeable gender difference occurred (Figure 1). The ratio was lower in women than in men before the operation (2.0 for women vs 2.9 for men, P=0.036, after correction with cell size P=0.099). However, after 1 y weight loss, the ratio increased markedly in women, reaching the level of 4.0 (P=0.028), whereas there was no change in men (2.7 after operation).

In women, the HSL mRNA level correlated positively with basal and maximal glycerol release in adipocytes before weight reduction (after correction with fat mass (kg): r=0.69, P=0.060 for basal release and r=0.76, P=0.029 for maximal release; after correction with subcutaneous cell size (pl): r=0.65, P=0.117 for basal release and r=0.77, P=0.045 for maximal release). In men, HSL mRNA level correlated positively with percent of body fat (r=0.79, P=0.059, and after correction for cell size r=0.89, P=0.044) and with cell size (r=0.76, P=0.029) 1 y after the operation.

Glycerol release in isolated adipocytes

Basal and maximally stimulated glycerol release values are shown in Table 3. In women, basal and maximal glycerol release did not change along with weight reduction. The relative change of basal glycerol release correlated strongly with the change in fasting serum glycerol concentration (r=0.971, P=0.006). Furthermore, there were no changes either in isoprenaline or adrenaline stimulated (data not shown) or UK or PIA inhibited glycerol release in isolated adipocytes (Figure 2) in women. However, the EC50 value of PIA for the women showed marked change decreasing from 30.5 µmol/l to the level of 3.8 µmol/l. The change in the EC50 value for UK was from 4.1 to 0.4 µmol/l. There was no change in the EC50 value for isoprenaline (before operation, 34.8 µmol/l; after 1 y weight reduction, 72.23 µmol/l).

In men, basal and maximal glycerol release decreased when calculated for cell size (Table 3). Furthermore, glycerol release stimulated by isoprenaline or adrenaline as well as corresponding EC50 values for isoprenaline (before, 51.7 µmol/l; after, 56.8 µmol/l) were similar in men both before and after the weight loss. The inhibition of isoprenaline stimulated glycerol release by UK or by PIA in isolated adipocytes improved after weight loss (P=0.001 and P=0.001, respectively; Figure 2). EC50 values for the men decreased from 1.2 to 0.6 µmol/l for UK and from 13.0 to 3.3 µmol/l for PIA.

There was no difference between genders in basal or maximal glycerol release either before or after the weight loss (Table 3). However, the relative change of the basal glycerol release differed markedly between genders. The difference in UK inhibited glycerol release between genders was shown (P=0.043, corrected for fat mass P=0.049) before weight reduction. The change in UK-induced inhibition was stronger in men than in women (P=0.059, corrected for fat mass P=0.049). PIA-induced inhibition differed between genders (P=0.046, corrected for fat mass P=0.206) after weight reduction. No gender differences in stimulation by isoprenaline or adrenaline either before or after 1 y weight reduction were found.

Microdialysis study

In women, isoprenaline stimulated glycerol release was also similar at both measurements (relative stimulation 3.3-fold (stimulated 10.4±2.0 pmol/µl, mean±s.e.m.) and 3.8-fold (stimulated 8.8±0.9 pmol/µl) before and 1 y after the operation, respectively). Furthermore, we did not find an inhibitive effect with any concentrations of adenosine or clonidine in women before or 1 y after weight loss.

In men, the stimulation by isoprenaline was also similar before (relative stimulation 2.3-fold, 10.0±2.2 pmol/µl) and after (2.6-fold, 5.2±0.6 pmol/µl) the weight loss. As in women, there was no inhibitive effect with adenosine or clonidine in men before the operation. However, an inhibition with 5 µmol/l adenosine was found after the weight loss (Figure 3), although the change was not statistically significant (P=0.116).

There was a difference between men and women (P=0.037) for the response to 5 µmol/l adenosine after the weight reduction due to the improved inhibition by adenosine observed in men.

The blood flow measured by ethanol clearance method showed stable blood flow in the adipose tissue in the site of the microdialysis during each infusion concentration of either isoprenaline, clonidine or adenosine. Blood flow increased, however, when the concentration of isoprenaline was increased from 10 to 30 mmol/l, but it stabilized again at the higher concentration. The basal blood flow ratio did not differ between genders either before (first probe, 0.92±0.13 for men, 0.90±0.21 for women; second probe, 0.83±0.07 for men, 0.77±0.07 for women; third probe, 0.92±0.14 for men, 0.98±0.11 for women) or after weight reduction (first probe, 0.87±0.05 for men, 0.84±0.11 for women; second probe, 0.71±0.07 for men, 0.86±0.11 for women; third probe, 0.65±0.05 for men, 0.83±0.05 for women).

Discussion

This study shows that adipose tissue metabolism and the expression of HSL and LPL in abdominal subcutaneous fat depot respond differently to weight reduction in men and women. In men neither HSL nor LPL mRNA expression changed, although the percentage of fat mass and the adipose cell size decreased along with weight reduction. Interestingly, however, weight loss improved the inhibition of lipolysis by adenosine analog and ₂-adrenergic agonist in isolated adipocytes and in the microdialysis study in men. In women, percentage of fat mass and adipose cell size decreased as in men, but in contrast to what was observed in men, the mRNA expression of HSL decreased in subcutaneous adipose tissue. This change was not, however, associated with major changes in adipose tissue metabolism in women. The observed gender difference cannot be ascribed to difference in the degree of obesity or changes in fat mass or fat cell size. To the best of our knowledge, gender differences have not been reported before between obese men and women in HSL expression along weight reduction, although many other aspects of adipose tissue metabolism have been studied and compared.^12,26,32,46

Previously, it has been found that HSL expression is a major determinant of maximal lipolytic capacity of the human fat cell and that is markedly affected by the cell size.^47,48,49 Furthermore, fat cell size has a great influence on the rate of lipolysis,⁵⁰ larger cells being lipolytically more active. Thus, there seems to be a tight association between cell size, HSL expression and lipolytic activity in human adipose tissue. However, controversial results have been reported when the response of HSL expression and activity to weight loss has been studied showing increase,²⁴ no change²² or even decrease²³ in women despite a marked reduction in adipose cell size. Discordant findings have also been reported concerning LPL expression along with weight loss.^10,11,12 Some studies have shown increase in LPL activity and mRNA expression in obese subjects during weight loss^10,11 and another, increase in activity, but no change in LPL mRNA expression in women, and no change in either of these parameters in men.¹² Furthermore, women have been shown to have higher LPL mRNA expression and activity in abdominal subcutaneous adipose tissue than men.²⁶ Underlining further the complexity of adipose tissue metabolism in obesity, contrasting findings have also been reported in respect of changes found in basal and maximal lipolysis in response to weight reduction in women.^22,23,24 This controversy might be partly explained by the difference in the stability of weight loss of subjects and, thus, the basal level of mobilization of fatty acids from adipose tissue at the time of the studies as well as by the difference in the study design. In a recent study,³² there was no change in basal lipolysis in either men or women, but enhanced lipolytic efficiency in response to weight loss in both genders was reported. This suggests reversibility of obesity caused changes in adipose tissue, since in earlier studies -adrenergic stimulation of lipolysis^14,28,51 has been found to be blunted in obesity. Weight loss has also been found to improve the inhibitory effect of adenosine in obese subjects,⁴⁰ although ₂-adrenergic inhibition or sensitivity to inhibition did not respond to moderate weight loss in men or women in the above-mentioned study.³²

In the present study, it was shown that women, in the state of morbid obesity, had a higher concentration of HSL mRNA in subcutaneous abdominal adipose tissue than men had. In women HSL mRNA expression associated with basal and maximal glycerol release before weight reduction. However, basal and maximal glycerol release did not differ between men and women. The difference in the cell size did not explain the difference in the expression in the present study. Along with weight reduction in men, no change in the level of HSL mRNA expression in subcutaneous abdominal adipose tissue was observed, whereas a clear decrease was observed in women. The decrease in HSL expression observed in women underlines the effect of decreased cell size due to weight loss. In men HSL expression remained at the same level after weight reduction, in spite of a decrease in fat cell size, and now, the association was observed between the level of expression and cell size. Thus, it seems that the response in HSL mRNA expression to weight reduction is markedly different in genders. In parallel with a decrease in HSL mRNA expression in women, a slight increase in LPL mRNA expression was observed, resulting in an increase in the LPL/HSL-ratio. It is possible that an improvement in insulin sensitivity,⁷ along with weight reduction, could participate in decreased HSL expression and in a slight increase of LPL expression observed in the adipose tissue from women. It should be noted, however, that HSL activation depends not only on the expression, but also on the phosphorylation state.⁴⁷ Thus, we cannot conclude whether or not the differences are also seen in HSL enzyme activity, although according to previous studies, HSL expression in adipose tissue gives actual information of protein and its activity.^24,47 Furthermore, LPL mRNA expression has been shown to parallel LPL synthesis.^10,26

Gender difference was also seen in lipolytic activity in abdominal adipose tissue along with weight reduction in the present study. In women, we did not find any major changes in the adipose tissue metabolism. In contrast, in men a decrease in basal and maximal lipolysis was found along with weight reduction and decreased cell size. The response to maximal stimulation was, however, unexpectedly low both before and after weight loss in both genders. In men, an improvement in ₂-adrenergic inhibition in vitro and in adenosine-inhibited lipolysis in vivo as well as in vitro was found. The finding was further confirmed by an improved sensitivity to UK and PIA at the group level in men. This indicates that the responsiveness and sensitivity to the regulation of lipolysis in adipose tissue was improved along with weight reduction. However, we did not find any change in responses of lipolysis to -adrenergic stimulation in either men or women.

It may be speculated that the partial discrepancy between our and previous studies might be due to differences in the stability of weight loss. The weight loss of our subjects had been greatly slowed down after the first 6 months, and most of the subjects can be considered having been in stable weight status at the time of the follow-up studies. The subjects were, however, still obese after 1 y weight loss, which may explain the unexpectedly low maximal stimulation of lipolysis. Obesity can cause attenuated responses in adipose tissue due to desensitization^28,52 or enhanced basal lipolysis so that responses to stimulation or inhibition before and after weight loss might be weaker than expected.

It can be speculated that a clear decrease in HSL mRNA and slight increase in LPL mRNA expressions in response to marked weight reduction in massively obese women might favor fat deposition. The unchanged basal and maximal lipolysis in women indicates, however, that they remained basal lipolytic capacity despite the decrease in cell size along with weight loss. In men, HSL as well as LPL mRNA expression remained unchanged, although basal and maximal lipolysis decreased, indicating that the expression level was not in straight relationship with the rate of lipolysis. Furthermore, in men, the changes in adipose tissue metabolism in vivo and in vitro show improved responsiveness and sensitivity to inhibition of lipolysis in adipose tissue. Thus, the present study is in line with previous findings about gender differences in adipose tissue metabolism and adds knowledge about this difference also regarding the responses to weight reduction.

Acknowledgements

This work was supported by the grants from Academy of Finland, Research Council for Health, Jenny and Antti Wihuri Foundation, Finland, Finnish Cultural Foundation of Northern Savo, Hoffman La Roche Ltd, Basel, Switzerland and EVO fund by Kuopio University Hospital. The authors also thank Mrs Paulette Vallier, Ms Teija Inkinen, Ms Erja Kinnunen, Mrs Kaija Kettunen, Ms Irja Lyytikäinen, Mrs Eeva Hakulinen and Mrs Sirkku Malila for skillful technical assistance and the nursing staff of the Operational Unit 1 and Surgical Ward 2205 of Kuopio University Hospital.

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Figure 1 Individual and mean (solid diamond) values for HSL mRNA expression in men (A), in women (B), LPL mRNA expression in men (C) and in women (D), LPL/HSL-ratio in men (E) and in women (F) before and after 1 y weight reduction. There were significant differences (P<0.05) in HSL mRNA expression and LPL/HSL-ratio before weight reduction between men and women.

Figure 2 Glycerol release in 10⁴ nmol/l isoprenaline stimulated subcutaneous abdominal adipocytes isolated from men inhibited with different concentrations of UK-14304 in men (A), N⁶-phenyl-isopropyladenosine (PIA) in men (B) UK-14304 in women (C), N⁶-phenyl-isopropyladenosine (PIA) in women (D) before and after 1 y weight reduction (**P<0.001 for difference in the responses before and after weight reduction in men); mean±s.e.m.

Figure 3 The effect of 1 and 5 µmol/l adenosine in the microdialysis study in men before and after 1 y weight reduction. The basal concentration of glycerol in dialysate and the average glycerol concentration of three successive 20 min fraction with 1 and 5 µmol/l adenosine are shown; mean±s.e.m.

Table 1 Age, anthropometric characteristics and cell size of subcutaneous abdominal adipocytes in men and women before and after 1 y weight reduction

Table 2 Biochemical characteristics of the subjects before and after 1 y weight reduction

Table 3 Basal and maximally stimulated lipolysis in men and women before and after 1 y weight reduction