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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1472-1480.)
© 1997 American Heart Association, Inc.

Articles

Sex Differences in Visceral Fat Lipolysis and Metabolic Complications of Obesity

Fredrik Lönnqvist; Anders Thörne; Valerie Large; ; Peter Arner

From the Departments of Medicine (F.L., V.L., P.A.) and Surgery (A.T.), Huddinge University Hospital, and the Research Center, Karolinska Institute, Stockholm (F.L., A.T., V.L., P.A.), Sweden.

	Abstract

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Abstract Cardiovascular complications of obesity are more common in men than women. Sex differences in visceral fat lipolysis may be of importance in this respect, since increased release of free fatty acids (FFAs) from visceral fat to the liver by the portal venous system has been thought to cause several metabolic complications due to obesity, such as hypertension, hyperlipidemia, and glucose intolerance. The aim of this study was to investigate sex differences in clinical characteristics and visceral fat mobilization in obesity. Obese subjects (22 male and 23 female) undergoing elective surgery were matched for body mass index and age. The males had both higher waist-to-hip ratio (WHR), sagittal diameter, blood pressure, fat-cell volume, plasma insulin, glucose, and triglyceride and lower HDL cholesterol levels than the females. The rate of norepinephrine-induced FFA and glycerol release was twofold higher in men (P=.02). No significant reutilization of FFA was observed. The difference in maximum norepinephrine-induced rate of lipolysis between men and women was independent of both WHR and sagittal diameter and was an independent regressor for levels of plasma glucose and plasma HDL cholesterol. Fat-cell volume was an independent regressor for plasma triglycerides and blood pressure. No sex differences in the lipolytic sensitivity to ß₁- or ß₂-adrenoceptor–specific agonists or in the antilipolytic effect of insulin were observed. However, the lipolytic ß₃-adrenoceptor sensitivity was 12 times higher (P=.004) and the antilipolytic ₂-adrenoceptor sensitivity 17 times lower (P=.003) in men. Furthermore, lipolysis induced by agents acting at the adenylate cyclase and protein kinase A levels were almost twofold enhanced in men. However, no sex difference in maximum hormone-sensitive lipase activity was observed. In conclusion, in obesity, catecholamine-induced rate of FFA mobilization from visceral fat to the portal venous system is higher in men than women. This phenomenon is partly due to a larger fat-cell volume but also to a decrease in the function of ₂-adrenoceptors, an increase in the function of ß₃-adrenoceptors, and an increased ability of cyclic AMP to activate hormone-sensitive lipase. These factors may contribute to gender-specific differences in metabolic and cardiovascular disturbances accompanied by obesity.

Key Words: adipocytes • free fatty acids • gender • adrenoceptors • ß₃-adrenoceptor • insulin • obesity

	Introduction

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Obesity is a major risk factor for the development of chronic disorders such as hypertension, non–insulin-dependent diabetes, dyslipidemia, and atherosclerosis, which in turn cause ischemic heart disease, stroke, and premature death.^{1 2 3} The prevalence of cardiovascular disease is greater in obese men than obese women,^{4 5} and the reason for this gender difference is not fully understood. Sex hormones are likely to be involved in the relative protection from cardiovascular disease in women before menopause.^{4 6} There are also distinct sex-dependent differences in the regional fat distribution. Susceptibility to upper-body fat accumulation, which occurs more frequently among men, has been shown to be more strongly associated with metabolic and cardiovascular diseases than is lower-body obesity.^{1 2 3} In contrast, gluteofemoral adipose tissue accumulation, which is more common in women, does not appear to increase substantially the risk of cardiovascular disease. It has therefore been proposed that when similar amounts of total body fat are present, obese men are at higher risk of developing secondary metabolic complications and cardiovascular disease than obese women.⁷ It has also been suggested that the sex differences in risk factors of cardiovascular disease to a great extent can be explained by the amount of visceral adipose tissue,⁸ which is larger in upper-body obesity.⁹ However, additional factors may also be involved.

A plausible explanation of the higher incidence of cardiovascular complications in upper-body obesity may be an increased secretion of free fatty acids (FFAs) produced by the visceral fat depots through lipolysis in this region.^{2 3} It has indeed been shown that obese subjects have a markedly higher FFA release from their visceral adipocytes than lean controls.¹⁰ The visceral fat depot has direct access to the liver through the portal venous system. Increased "portal" FFA may have a number of adverse effects on the liver, such as an increase in gluconeogenesis, impairment of metabolism and action of insulin, and increased lipoprotein synthesis.^{2 3 11 12} These may in turn contribute to the development of glucose intolerance, hypertension, dyslipoproteinemia, and atherosclerosis.

Lipolysis in humans is above all regulated by insulin, which inhibits lipolysis and catecholamines. The catecholamine effects are modulated through four adrenoceptor subtypes, ie, stimulation via ß₁-, ß₂-, and ß₃-adrenoceptors and inhibition via ₂-adrenoceptors.¹³ Insulin and catecholamine receptors regulate cyclic AMP formation, which in turn modulates hormone-sensitive lipase, the enzyme that turns on lipolysis.

In vivo data suggest that lipolysis regulation is different in lower-body obesity than in upper-body obesity.^{14 15 16} Whether a gender difference in FFA mobilization from the visceral fat depots exists remains to be established, and if it does exist, the possible underlying mechanisms need to be explored.

The present study was performed to investigate possible sex differences in clinical characteristics and visceral FFA mobilization through lipolysis in obesity. Gender differences may be localized at any step in the chain of events that transfers the signal from the hormone receptors to the hormone-sensitive lipase. Consequently, we have investigated catecholamine- and insulin-induced lipolysis in visceral (omental) fat cells in obese men and women.

	Methods

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Patients
The study comprised 22 male and 23 female Scandinavian obese subjects with average body mass index (BMI) of 34±1 kg/m², range 29 to 56, undergoing laparoscopic cholecystectomy or weight-reduction surgical treatment through open or laparoscopic procedures at Huddinge University Hospital. The two groups were matched for age, BMI, and smoking habits and were clinically characterized before surgery. Except for obesity and/or gallstone disease, they had no apparent diseases and took no medication. The age of the males ranged from 24 to 55 years and of the females from 24 to 50 years. All women included in the study had stated that they had regular menstruations. Furthermore, FSH was analyzed in the nine women that were over 40 years old. The data show that three of the women were perimenopausal (FSH 36 to 42 IU/L) and the rest of the women were clearly premenopausal, with FSH values ranging from 2 to 17 IU/L. The waist-to-hip ratio (WHR), the sagittal diameter, and the systolic and diastolic blood pressures were measured in the supine position on the day before surgery. The WHR in the female group ranged from 0.89 to 1.08 and from 0.97 to 1.12 in the male group, and the sagittal diameter ranged from 24 to 36 cm in women and from 25 to 37 cm in men. The sagittal diameter was obtained by measuring the distance from the examination table to a horizontal crossbar placed over the abdomen of a recumbent subject at the crista level. Blood pressure was measured with a mercury sphygmomanometer on the right arm. The systolic and diastolic pressures were determined by using phases I and V of the Korotkoff sounds, the values being the means of three consecutive measurements after a 10-minute rest. After an overnight fast, the study subjects were still resting in bed for 15 minutes when venous blood samples were obtained for determination of metabolic laboratory parameters. They were all analyzed by the hospital's routine chemistry laboratory, except for insulin, which was measured with a radioimmunoassay kit (Pharmacia).

General anesthesia was induced at 8 AM by a short-acting barbiturate and maintained by fentanyl and a mixture of oxygen and nitrous oxide. Intravenous saline was administered before the fat biopsies were taken from the major omentum at the beginning of the operation.

The study was approved by the ethics committee of Karolinska Institute, Stockholm. All patients gave informed consent to participate in the study.

Isolation of Fat Cells and Determinations of Cell Size and Number
For technical reasons we could not obtain specimens larger than 0.3 to 1.0 g of omental fat during laparoscopic cholecystectomy. The weight-reduction surgery was initiated by using laparoscopic technique in about half of the cases, yielding small adipose tissue biopsies. The adipose tissue was immediately transported to the laboratory in saline at 37°C and isolated fat cells were prepared from the fat specimens by collagenase treatment, as described by Rodbell.¹⁷ The cells were kept in an albumin solution, as described below, and the cell density of the fat-cell suspension was maintained constant by slow stirring with the aid of a magnet. Direct microscopic determination of the fat-cell diameter, performed according to the method of Di Girolamo and coworkers,¹⁸ was calculated by using 200 cells from each subject. The mean fat-cell volume and weight were determined, taking into account the skewness in the distribution of the cell diameter and using the method described by Hirsch and Gallian.¹⁹ The total lipid content of each incubation was determined gravimetrically after organic extraction. Assuming that lipids constitute >95% of the fat-cell weight, the number of fat cells can be calculated by dividing the total lipid weight by the mean cell weight. This indirect method of determining the fat-cell number was compared with a tedious direct method,²⁰ in which all cells are counted in appropriately diluted cell suspensions. The two methods gave almost identical results (r=.97) in 10 consecutive experiments, using linear regression analysis.

Lipolysis and Antilipolysis Experiments
Diluted suspensions (0.2 mL) of isolated fat cells (5000 to 10 000/mL) were incubated in duplicate for 2 hours with or without increasing concentrations of either norepinephrine; the nonselective ß-adrenergic agonist isoprenaline; the selective ß₁-adrenoceptor agonist dobutamine; the selective ß₂-adrenoceptor agonist terbutaline; the selective partial ß₃-adrenoceptor agonist CGP 12177;²¹ forskolin, which acts at the catalytic component of the adenylate cyclase; the nonhydrolyzable cyclic AMP analogue dibutyryl cyclic AMP,²² which acts on protein kinase A; or the selective ₂-adrenoceptor agonist UK 14304.²³ All incubations were performed at 37°C in Krebs-Henseleit phosphate buffer (pH 7.4), supplemented with glucose (1 g/L), bovine serum albumin (20 g/L), and ascorbic acid (0.1 g/L), with air as the gas phase. The ligands were added simultaneously at the start of the incubation. The concentration range used for each drug depended on its lipolytic performance. Overall, it ranged from 10^-12 to 10^-3 mol/L. The same batches of collagenase and albumin and the same stock solutions of adrenoceptor agonists were employed throughout the study. As discussed in detail previously,²⁴ adenosine leaking out from isolated fat cells may interfere with the ₂-adrenoceptor–mediated antilipolytic effect of catecholamines. In our dilute incubation system, there is minimal influence of adenosine contamination. However, in the ₂-adrenoceptor experiments with UK 14304, adenosine deaminase (1 mU/mL) was added to prevent antilipolytic interactions with traces of adenosine that might still be present and induce additional inhibitory effects²⁴ in the diluted cell suspensions. The influence of adenosine on lipolysis in a fat-cell system like ours is, on the other hand, negligible when adipocytes are stimulated with lipolytic drugs, as previously discussed.²⁵ Therefore, we preferred not to add adenosine deaminase in the remaining concentration-response experiments. In the experiments with UK 14304, the incubation medium was also supplemented with 10^-3 mol/L 8-bromo cyclic AMP to increase the initial (basal) rate of lipolysis, which was too low in visceral fat cells to be inhibited by UK 14304. We have previously shown that 8-bromo cyclic AMP–induced lipolysis can be fully inhibited by antilipolytic agents in human fat cells.²² There was no difference in sensitivity to 8-bromo cyclic AMP between obese and nonobese subjects. The glycerol concentration after the 2-hour incubation was determined in a cell-free aliquot by a bioluminescence method.²⁶

The insulin experiments were designed to estimate the antilipolytic effect of various insulin concentrations, ranging from 10^-13 to 10^-9 mol/L, on lipolysis induced by norepinephrine in increasing concentrations from 10^-12 to 10^-4 mol/L. Norepinephrine was preferred as lipolytic inducer in these experiments because it is the natural lipolytic agent. Methodological experiments revealed that a maximum antilipolytic effect was always obtained with 10^-9 mol/L of insulin.

All agonists caused a concentration-dependent stimulation or inhibition of glycerol release that reached a plateau at the highest agonist concentrations. Concentration-response curves for glycerol release were used to determine the concentrations of the adrenoceptor agonists, giving half of their own maximum stimulation (ß) or inhibition (₂). These EC₅₀ values (expressed as log mol/L) were determined by linear regression analysis of log-logit transformation of the ascending (ß agonists) or descending (₂ agonists and insulin) part of the individual concentration-response curves. The EC₅₀ value reflects specific agonist-adrenoceptor interactions, since at this concentration the selective agonist has few if any interactions with other adrenoceptor subtypes. Lipolysis rates in the absence of, or in the presence of, maximum effective agonist concentrations were related to fat-cell number. The maximum lipolytic effects indicate agonist responsiveness. Since the amount of adipose tissue available was limited because of laparoscopic surgery, we were not able to perform the antilipolysis experiments with UK 14304 and insulin in all subjects. The experiments with UK 14304 were performed on 10 women and 8 men, while the insulin experiments were performed on 13 women and 11 men. In both cases, the male and the female groups were matched for age and BMI.

Simultaneous Determination of Glycerol and FFA Release
The fat-cell isolation and incubation procedures were exactly the same in these experiments as in the lipolysis experiments described above, except that fatty acid–free bovine serum albumin 0.25% (wt/vol) was used as an acceptor of FFA in the incubation medium. At the end of the incubation, one aliquot of the medium was removed for glycerol determination as described above. Another aliquot was used to measure FFA release. The method for determining FFA has been described in detail.²⁷ In brief, the FFA analyses were performed as follows. The assay involved pretreatment with the detergent SDS to liberate the FFAs from the bovine serum albumin before the activation of fatty acids by acyl-CoA synthetase. This step was followed by oxidation of the resulting thioesters by acyl-CoA oxidase. The H₂O₂ formed (reflecting the amount of FFA) was subsequently measured in a horseradish peroxidase–catalyzed luminol reaction, using a luminometer (LKB Wallac). Lack of adipose tissue reduced the simultaneous determinations of FFA and glycerol release to 30 of the 45 subjects (18 women and 12 men).

Assay of Hormone-Sensitive Lipase Activity
Aliquots of 300 µL of isolated adipocytes were homogenized in 3 mL of a buffer containing 0.25 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and the protease inhibitors leupeptin and antipain, both at 20 µL/mL. The samples were then centrifuged at 100 000g for 45 minutes at 4°C in a Beckman ultracentrifuge L8-60M. The fat-free infranatant was recovered for analysis of enzymatic activity, which was determined using 1(3)-mono(³H)oleoyl-2-oleoylglycerol as substrate. All samples were incubated in triplicate on one occasion for 30 minutes at 37°C. The use of a diacylglycerol analogue as substrate enhances the sensitivity of the assay, since hormone-sensitive lipase has a 10-fold higher activity toward diacylglycerol than triacylglycerol.²⁸ Moreover, since this substrate has only one hydrolyzable ester bond at the 1(3)- position, neither the substrate itself nor its hydrolysis products can be hydrolyzed by monoacylglycerol lipase, which is abundant in adipose tissue. Furthermore, under the conditions for the assay, ie, pH 7.0 and absence of apolipoprotein C-II, very low lipoprotein lipase activity is measured.²⁸ One unit of enzyme activity is defined as 1 µmol of fatty acid release per minute at 37°C. Lipase activity was related to fat-cell number. This value was calculated by dividing the volume of cells homogenized by the fat-cell volume of each subject. Determination of hormone-sensitive lipase activity required large amounts of tissue and was therefore performed in a subgroup of 8 male and 12 female subjects. The males and females were matched for age and BMI.

Drugs and Chemicals
Bovine serum albumin (fraction V, lot 63F-0748), fatty acid–free bovine serum albumin (lot A-6003), Clostridium histolyticum collagenase type I, glycerol kinase from E. coli (G4509), acyl-CoA synthetase (EC 6.2.1.3) from Pseudomonas species, acyl-CoA oxidase from Candida lipolytica, horseradish peroxidase (EC 1.11.1.7, Type VI; 250 to 330 U/mg), SDS, ascorbate oxidase, adenosine deaminase, Triton X-100, ATP, forskolin, dibutyryl cyclic AMP, and 8-bromo cyclic AMP were obtained from Sigma Chemical Company. Norepinephrine and (-)-isoprenaline hydrochloride came from Hässle, terbutaline sulfate from Draco, dobutamine hydrochloride from Lilly, UK 14304 tartrate from Pfizer, CGP(±)12177 [(-)-4-(3-t-butylamino-2-hydroxy-propoxy)benzimidazole-2-one] from Ciba Geigy, and human insulin from Novo-Nordisk. ATP monitoring reagent containing firefly luciferase came from LKB Wallac. Antipain and leupeptin were obtained from Sigma. 1(3)-Mono(³H)oleoyl-2-oleoylglycerol was manufactured in the Department of Medical and Physiological Chemistry, Lund University, Sweden. All other chemicals were of the highest grade of purity commercially available. Collagenase and other ingredients in the buffers came from the same batches throughout the study.

Statistical Analysis
Values are given as the mean±SEM. EC₅₀ values were logarithmically transformed to normalize the data and were defined as ß- and ₂-adrenoceptor subtype sensitivity, respectively. ANCOVA, ANOVA, the Student's two-tailed unpaired t test, and stepwise regression analysis were employed for statistical comparisons. All analyses were performed with a statistical software package.

	Results

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The clinical characteristics in obese men and women are shown in Table 1. As expected, the mean WHR and sagittal diameter were significantly higher in the obese men than in the obese women, despite similar BMI values in the two groups. Likewise, the sagittal diameter and the omental fat-cell volume were higher in obese men, who also had higher diastolic blood pressure, plasma insulin, glucose, and triglyceride levels and lower HDL cholesterol levels than the female group. The majority of the men had an upper-body distribution of the fat mass, with several signs of classical complications of obesity, including indirect evidence of insulin resistance.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics in Obese Men and Women

The effects of increasing concentrations of norepinephrine on the simultaneous release of glycerol and FFA from omental fat cells in obese men and women are depicted in Fig 1, in which basal lipolysis has been subtracted. The male group had an almost twofold higher visceral fat FFA and glycerol response to norepinephrine than the female group (repeated two-way ANOVA, P=.02 and P=.02, respectively). Also, at physiological concentrations of norepinephrine (1 to 10 nmol/L), the male group demonstrated statistically significant 50% to 100% higher norepinephrine-induced glycerol release rates than the female group. At 10 nmol/L of norepinephrine, the lipolysis rates were 14.8±1.7 and 7.9±1.3 µmol glycerol per 10⁷ cells per 2 hours in men and women, respectively (P=.0002). The differences were even larger if basal lipolysis was not subtracted. The individual glycerol release-to-FFA release ratios were calculated on net lipolysis (minus basal lipolysis) at 10^-7 mol/L of norepinephrine when there was a maximum rate of FFA release in both groups. The glycerol:FFA ratios were not significantly different (1:2.93±0.17 and 1:2.69±0.18 in men and women, respectively). Thus, in none of the groups was there an important reesterification of FFA.

View larger version (13K): [in this window] [in a new window]   Figure 1. Simultaneous release of glycerol and FFA from fat cells. Omental fat cells from obese men (A) and obese women (B) were incubated with increasing concentrations of norepinephrine. Release of FFA () and glycerol () are shown without basal lipolytic activity. The results are mean±SEM. The groups were compared by ANOVA. The F and probability values for FFA and glycerol, respectively, are given.

It was also important to elucidate whether the gender difference in FFA and glycerol mobilization from the visceral fat depot was simply due to differences in omental fat-cell size. As the obese men had larger omental fat-cell volumes, the lipolytic rates were therefore also calculated in relation to the cell-surface area, to adjust for this difference. However, the gender-specific differences in maximum norepinephrine-induced lipolysis remained after this correction and were 1.93±0.15 and 1.46±0.16 µmol glycerol per square micrometer x10⁴ cells (P=.04), respectively. Furthermore, lipolysis was investigated in a subgroup of subjects matched for fat-cell volume. All female and male subjects with a fat-cell volume of <450 pL or >800 pL were excluded. The remaining 16 men and 16 women had similar mean fat-cell volumes (684±30 versus 642±41 pL), and the mean age and mean BMI values were almost identical. In spite of this adjustment, the differences in maximum norepinephrine-induced lipolysis between men and women remained; values were 24.9±2.5 versus 15.8±1.9 µmol glycerol per 10⁷ cells per 2 hours (P=.009) in men and women, respectively. No significant relationships were seen between adipocyte cell size and maximum norepinephrine-induced lipolysis in either sex. However, adipocyte cell size was correlated to basal lipolysis in women (r=.51, P=.02).

Finally, the all-over norepinephrine-induced lipolytic rate from the visceral fat-cell mass was also calculated in both sexes. First, maximum norepinephrine-induced lipolysis rates were 3.63±0.28 and 3.21±0.39 µmol glycerol per gram triglyceride in men and women, respectively. From the sagittal diameter, the volumes of intra-abdominal fat can be calculated.²⁹ The visceral fat depots constitute about 80% of the total intra-abdominal fat mass and were calculated to be 10.1±0.4 L in men and 4.8±0.2 L in women. This means that men in general have not only larger fat cells but also more visceral fat cells than women. From the anthropometric measurements, the number of visceral fat cells could be calculated to 12.6x10⁹ and 5.9x10⁹ in men and women, respectively. By combining these measurements, maximum rate of norepinephrine-induced lipolysis was calculated to be 367±28 mmol glycerol per 2 hours in men and 189±23 mmol glycerol per 2 hours in women (P=.0001).

The markedly higher catecholamine-stimulated glycerol and FFA release in the obese men can be attributed to a difference in any one of the stimulatory ß₁-, ß₂-, and ß₃-adrenoceptors, the inhibitory ₂-adrenoceptors, or in postreceptor function. All these possibilities were therefore investigated by incubation of fat cells in the presence or absence of specific agents acting at different steps in the lipolytic cascade. Glycerol release was determined and used as a lipolytic index.

Fig 2 demonstrates the lipolytic and antilipolytic sensitivities of omental fat cells to selective ß- and ₂-adrenoceptor agonists, ie, dobutamine (ß₁), terbutaline (ß₂), CGP 12177 (ß₃), and UK 14304 (₂) in men and women. The mean concentration-response curves for all four adrenoceptor agonists are given as a percent of their maximum effect, to elucidate differences in agonist sensitivity between groups (ie, leftward or rightward shift in the concentration-response curve). There were no apparent differences in ß₁- or ß₂- adrenoceptor sensitivity between the two groups. However, omental fat cells of obese men were 12 times more sensitive to the ß₃-adrenoceptor agonist CGP 12177 (P=.004) and also demonstrated a 17 times lower ₂-adrenoceptor sensitivity after stimulation with UK 14304 (P=.003). From the concentration-response curves, the mean EC₅₀ values (log mol/L) for stimulation or inhibition with the four different adrenoceptor agonists were calculated. The data are given in Table 2. The sensitivity to CGP 12177 and UK 14304 differed between men and women by at least 1 log unit (P<.01), but there was no sex difference as regards sensitivity to terbutaline or dobutamine.

View larger version (26K): [in this window] [in a new window]   Figure 2. Mean concentration-response curves for selective adrenergic agonists. Omental fat cells from obese men () and obese women () were incubated with increasing concentrations of selective adrenoceptor agonists, ie, dobutamine (ß1; A), terbutaline (ß2; B), CGP12177 (ß3; C), or UK 14304 (2; D). The adrenoceptor effect is expressed as percent of the maximum stimulatory or inhibitory effect. Values are mean±SEM. Individual EC50 values derived from these experiments were calculated for each of the agonists. The sensitivities in men and women were compared by the Student's t test. The EC50 data (expressed as log mol/L) are given in Table 2.

View this table: [in this window] [in a new window]   Table 2. Lipolytic Sensitivity of Omental Fat Cells to Adrenergic Agonists in Obese Men and Women

The mean values for basal lipolysis and the lipolytic response induced by the individual maximum effective concentrations of norepinephrine, isoprenaline, and the postreceptor-acting agents forskolin and dibutyryl cyclic AMP in men and women are depicted in Fig 3. Lipolysis was almost twice as high at both the receptor level and the two postreceptor steps examined (P=.002 to .0001). The maximum ₂-adrenoceptor–mediated antilipolytic response, about 50% inhibition, did not differ between the genders (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 3. Maximum lipolytic response induced by selective ß-adrenergic agonists or agents acting at the postreceptor level. Omental fat cells from obese men (solid bar) and obese women (open bar) were incubated in the absence or presence of isoprenaline (ISO), norepinephrine (NE), forskolin, and dibutyryl cyclic AMP (dcAMP). The lipolytic responses at maximum-effective concentrations were determined after subtraction of basal lipolysis. The maximum-effective concentrations varied intraindividually and were as follows: isoprenaline 10-9 to 10-5 mol/L, norepinephrine 10-8 to 10-6 mol/L, forskolin 10-7 to 10-5 mol/L, and dibutyryl cyclic AMP 10-3 to 10-2 mol/L. The groups were compared by the Student's t test; values are mean±SEM. The probability values for the statistical differences between men and women are given.

It was necessary to evaluate whether sex differences in lipolysis were influenced by other factors. Therefore we investigated the relative importance of age, sex, sagittal diameter, and plasma insulin for basal lipolysis, maximum norepinephrine-induced lipolysis, ß₃-adrenoceptor sensitivity, ₂-adrenoceptor sensitivity, and fat-cell volume, respectively, in men and women together by two-way stepwise regression analyses. The variable with the highest partial correlation coefficient was entered at each step until no variable remained with an F value of 4 or more. A summary of the results is depicted in Table 3. As the two groups were matched for age and BMI, these parameters were unlikely to be of importance for the gender-specific differences in lipolysis in this study. Gender was the only factor that contributed significantly to the variations in norepinephrine-induced maximum lipolysis, ß₃-adrenoceptor sensitivity, and ₂-adrenoceptor sensitivity between individuals. Gender was entered as the first and only step in all three cases, as indicated in Table 3. Gender seemed to be most important for the ₂-adrenoceptor function (adjusted r=.72) but also influenced ß₃-adrenoceptor sensitivity and maximal lipolysis considerably (adjusted r=.42 and .54, respectively). The sagittal diameter, which we used to measure the amount of visceral fat, was not associated with these four parameters. Nor was the WHR of importance in this respect (data not shown). However, sagittal diameter was entered as the first and only step when we investigated the contribution of the same clinical factors for the individual variations in fat-cell volume (adjusted r=.53). Also, WHR contributed significantly to the variations in fat-cell volume (data not shown).

View this table: [in this window] [in a new window]   Table 3. Importance of Clinical Characteristics for Lipolytic Function in Obese Men (n=22) and Women (n=23), Investigated by Stepwise Regression Analysis

We also investigated whether lipolysis was correlated with the clinical characteristics presented in Table 1. Therefore, the importance of age, sex, sagittal diameter, maximal lipolysis, and fat-cell volume for levels of plasma glucose, insulin, HDL cholesterol, triglycerides, and diastolic blood pressure were also investigated for the whole study sample, using stepwise regression analysis (Table 4). Several significant associations between lipolysis and clinical characteristics were observed. The norepinephrine-induced lipolysis rate at the maximum effective concentration contributed together with gender to plasma glucose (adjusted r=.53) and was entered as the first and only step when the contribution to HDL cholesterol variation was investigated (adjusted r=.38). On the other hand, sex was the only factor that contributed to the variations in plasma insulin levels (adjusted r=.44), while fat-cell volume was the only factor that contributed to the variations in plasma triglycerides (adjusted r=.34) and also contributed together with age to diastolic blood pressure (adjusted r=.57). Multiple regression analysis was also performed with the same variables in the model as in the stepwise analysis. Multiple regression gave similar results as stepwise regression (data not shown).

View this table: [in this window] [in a new window]   Table 4. Importance of Gender, Age, Sagittal Diameter, Fat-Cell Volume, and Maximum Lipolysis Rate for Clinical Characteristics in Obese Men (n=22) and Women (n=23), Investigated by Stepwise Regression Analysis

Since the lipolysis data also indicated a difference between genders at the postreceptor level, hormone-sensitive lipase activity was measured in a subgroup of 8 male and 12 female subjects, matched for age and BMI. No statistical difference in hormone-sensitive lipase activity was seen between the groups. The values were 15.5±3.7 mU/10⁷ cells and 12.3±6.3 mU/10⁷ cells in men and women, respectively.

The possible influence of gender on the ability of insulin to inhibit norepinephrine-induced lipolysis was also investigated. Since the rate of norepinephrine-induced lipolysis was sex dependent, it was necessary to match the initial rate of lipolysis (ie, no insulin present) in men and women before measuring the antilipolytic effect of insulin. Norepinephrine concentrations of 10^-8 mol/L in male fat-cell experiments and 10^-7 mol/L in female fat experiments were found to induce similar lipolytic rates (17.5±2.9 and 18.3±2.5 µmol glycerol per 10⁷ cells per 2 hours in men and women, respectively). These norepinephrine concentrations were therefore used to compare the antilipolytic effects of insulin. The similarity in lipolysis rates between the sexes at these two norepinephrine concentrations can also be observed in Fig 1. Fat cells were incubated in the absence or presence of insulin in concentrations ranging from 10^-14 mol/L to 10^-9 mol/L, with 10^-8 mol/L of norepinephrine present in the experiments performed on omental adipocytes from men and 10^-7 mol/L in the experiments performed on omental adipocytes from women. As demonstrated in Fig 4, no significant differences in insulin sensitivity or in the maximal antilipolytic effect of insulin (ie, at 10^-9 mol/L) between men and women were found. Insulin inhibited lipolysis by about 50% in both groups, at 10^-9 mol/L.

View larger version (17K): [in this window] [in a new window]   Figure 4. Antilipolytic effect of insulin in obese men and women. A comparison of the antilipolytic effect of insulin, added in increasing concentrations, on norepinephrine-stimulated lipolysis is made between obese men and women. Values are mean±SEM. A two-way ANOVA was used for statistical comparisons. No sex difference in insulin activity in the visceral fat was observed.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that in obesity the rate of FFA mobilization from the visceral fat cells is much higher in men than in women. Norepinephrine stimulation of visceral fat cells in males resulted in twofold higher average FFA and glycerol responses than in obese women. The glycerol to FFA ratio was near 1:3 in both genders, arguing for a minimal level of reesterification of FFA during catecholamine stimulation, which in turn indicates that the higher catecholamine-induced FFA mobilization in obese men is due to an increased lipolytic response. However, there was no sex difference in the antilipolytic effect of insulin. The results of regression analysis revealed that the sex differences in lipolysis demonstrated in this study could not be solely explained by variations in fat-cell volume and/or visceral fat mass. Although the WHR and sagittal diameter were higher in men, the contribution of gender to lipolysis variations was independent of these measures.

Sex-dependent differences in lipolytic function have previously been demonstrated. Women have a similar ß-adrenoceptor sensitivity and responsiveness but a lower ₂-adrenoceptor sensitivity in subcutaneous abdominal fat than men have, both in obese³⁰ and nonobese subjects.³¹ It has been suggested that the ₂-adrenergic component found in omental adipocytes of massively obese women may represent a protective mechanism against the metabolic complications associated with obesity.³² The fact that women also display lower rates of basal and norepinephrine-stimulated visceral fat-cell lipolysis than men³³ supports the hypothesis that a lower lipolytic activity in this fat depot may be beneficial. In addition, gender differences in fat-cell size and lipoprotein lipase activity have been demonstrated in morbidly obese patients.³⁴ Furthermore, differences in lipolysis between men and women have been reported from studies of visceral adipose tissue in nonobese subjects, showing that premenopausal and perimenopausal women have a smaller fat-cell size, lower glycerol release, and lower lipoprotein lipase activity than men.³⁵ On the other hand, another study of visceral fat from massively obese subjects showed no significant differences between the sexes in any of these parameters.³⁶ The discrepancy between this latter report and our own study could be explained by several factors. First, only the maximum lipolytic responses to norepinephrine were studied previously; no concentration response experiments were performed. Second, a 50% higher norepinephrine-stimulated maximum glycerol release in obese men than in obese women was also observed in the previous study, although the difference was not significant. Third, the study groups in this former report were not as large, and finally, glycerol release was expressed per cell-surface area instead of per number of fat cells. However, even if we adjusted for the gender-specific differences in fat-cell size by expressing lipolysis per cell-surface area, the difference in lipolysis between men and women remained. Also, when two subgroups of men and women with similar fat-cell volumes were compared, the gender-specific difference remained. Thus, most information obtained supports the idea that FFA mobilization is influenced not only by fat-cell size but also by gender itself.

We also estimated rates of lipolysis and total fat-cell number in the visceral fat depot using the sagittal diameter. We are aware that sagittal diameter is a rough estimate of total visceral fat in obese humans.³⁷ However, taking into consideration the limitations with the calculation, obese men mobilized lipids at approximately a double rate and also had twice as many fat cells in their visceral depot compared with obese women. In other words, a mass effect (larger total fat depot and more fat cells) might contribute to the larger FFA mobilization to the portal venous system in obese men compared with obese women.

The mechanisms underlying the marked sex differences in lipolytic response to norepinephrine found in this study could be partly revealed. The difference was observed when lipolysis was stimulated at various levels (ß-adrenoceptors, adenylate cyclase, and protein kinase A). However, the maximum hormone-sensitive lipase activity rates were similar in men and women. These observations indicate that in fat cells of obese male subjects, cyclic AMP must have a more pronounced ability to activate the hormone-sensitive lipase complex. This could be located at the level of protein kinase A or the phosphorylation/dephosphorylation of the hormone-sensitive lipase. Unfortunately, the amount of tissue available was far too small to explore such events.

We observed marked sex differences in ß₃- and ₂-adrenoceptor sensitivities. An almost 20-fold lower antilipolytic ₂-adrenoceptor sensitivity was demonstrated in obese men. Since the amounts of adipose tissue available were limited, it was possible to obtain ₂-adrenoceptor function data from only about half of the subjects. For the same reason, we could not investigate ₂-receptor binding and interactions with the so-called G-proteins. In contrast to ₂-adrenoceptors, no previous comparisons between sexes have been performed concerning ß₃-adrenoceptor function. However, it has been shown that visceral fat cells in obese subjects have increased sensitivity to ß₃-adrenoceptor stimulation and increased FFA release.¹⁰ The reason for the higher ß₃-adrenoceptor sensitivity in obese men observed in the present study is unknown. Unfortunately, no technique to quantify mRNA and protein for the ß₃-adrenoceptor in human fat cells was available at the time when this study was performed. Sex was an independent regressor for the variations in both ₂- and ß₃-adrenoceptor sensitivity in this study. Most probably the differences in antilipolytic ₂-adrenoceptor function and lipolytic ß₃-adrenoceptor function contribute to the sex differences in norepinephrine-induced lipolysis.

The clinical relevance of the sex differences in visceral fat lipolytic function demonstrated in the present analysis should also be considered. None of the patients included in the current study had any overt disease, apart from obesity. However, the obese men demonstrated as a group more marked signs of the insulin resistance or metabolic syndrome. As expected, anthropometric measurements showed that the men also had a more marked upper-body fat distribution, although such measures may be less accurate in massively obese subjects, as discussed in detail recently.³⁸ Furthermore, the obese men had more marked hyperinsulinemia, glucose intolerance, dyslipidemia, and higher blood pressures, which are all factors resulting in an increased risk of cardiovascular disease.^{1 2 3} Could the augmented lipolysis in visceral fat explain some of the complications observed in our obese subjects? It has previously been demonstrated in men that catecholamine sensitivity and responsiveness is higher in omental than in subcutaneous adipocytes and that glucose and insulin levels correlate more strongly with the visceral than the subcutaneous fat mass.³⁹ The results of stepwise regression in the present study support these data and indicate that not only enlarged fat cells and augmented lipolysis in visceral fat but also gender itself may contribute to metabolic complications of obesity. Therefore, not only hypertrophy but also hyperhydrolysis of the omental fat cells seems to influence the metabolic profile. Thus, lipolysis in omental fat cells contributed independently to the variations in the levels of plasma glucose and HDL cholesterol. One might therefore speculate that the impairment in glucose and lipid metabolism in obese male compared with obese female subjects could be due in part to increased FFA release from visceral fat in the males. However, it should be emphasized that this theory is based on in vitro lipolysis studies. Unfortunately, it is impossible for ethical reasons to investigate visceral lipolysis in vivo in humans.

We believe that both fat-cell size and the total visceral fat volume influence FFA mobilization and metabolic complications of obesity. However, the present data demonstrate that gender and maximum lipolytic rate are also important factors in this respect. In particular, blood glucose and plasma HDL cholesterol seem to be influenced by visceral adipocyte FFA mobilization. Therefore, the clinical findings in this study to some extent contrast with several previous reports indicating that sex differences in serum lipids, glucose and insulin, blood pressure, and coronary heart disease could be mainly explained by the variations in WHR and intra-abdominal fat mass.^{7 8 40 41 42} For example, adjustment for WHR reduced the sex differences in triglycerides.⁴⁰ Such conclusions were less clear for HDL cholesterol.⁴¹ However, all these studies have primarily been performed in nonobese or moderately obese subjects. Furthermore, mainly upper-body obese men were previously compared with mainly lower-body obese women. In this study upper-body obesity predominated in both groups. Finally, the temporal relations of body fat distribution to metabolic derangements are unclear. It should also be emphasized that the tendency to accumulate intra-abdominal fat is not only a sex-linked phenomenon but also a marker of genetic, hormonal, or lifestyle factors that may be directly associated with coronary heart disease and its risk factors. Even after considering relative weight, decreases in skeletal muscle sensitivity to insulin and hepatic removal of insulin occur in women with abdominal obesity.⁴³ Thus, there are several mechanisms whereby both the deposition of adipose tissue and the regulation of the intra-abdominal lipolytic rate could result in metabolic or other disturbances in obesity.

In conclusion, we suggest that the higher risk of metabolic derangements in obese men than in obese women may be caused by a larger visceral fat mass and an almost twice as high norepinephrine FFA release from the intra-abdominal fat cells to the portal venous system. If one considers these two factors together, the difference in FFA release per total visceral fat mass in obesity may be even greater in men than women compared with predictions from lipolysis data alone. The mechanisms underlying the findings concerning lipolysis could be a higher ß₃-adrenoceptor sensitivity, a lower ₂-adrenoceptor sensitivity, and an enhanced ability of cyclic AMP to activate hormone-sensitive lipase in visceral fat cells of obese men.

	Acknowledgments

 
This study was supported by grants from the Swedish Medical Research Council (B91-19F-9390-01), Karolinska Institute, the Swedish Society of Medicine, the Swedish Diabetes Association, the Nordic Insulin Foundation, Parke-Davis, and the foundations of Åke Wiberg, Tore Nilsson, Torsten and Ragnar Söderberg, Swedish Heart and Lung, King Gustav V, and Queen Victoria and Einar Belvén Golje. The excellent technical assistance of Britt-Marie Leijonhufvud, Catharina Sjöberg, Eva Sjölin, and Kerstin Wåhlin is greatly appreciated.

	Footnotes

 
Reprint requests to Peter Arner, MD, PhD, Department of Medicine, Huddinge University Hospital, 14186 Huddinge, Sweden.

Received December 29, 1995; accepted August 5, 1996.

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