Abstract
Objective: The aim was to investigate the impact of polycystic ovary syndrome (PCOS) on the regulation of lipolysis by catecholamine and for the first time atrial natriuretic peptide (ANP) before and after 16 wk of aerobic training.
Patients: Eight hyperandrogenic obese women with PCOS [age, 25 ± 1 yr; body mass index (BMI), 32.0 ± 1.6 kg/m2] and seven healthy BMI-matched controls participated. Studies were performed before and after a 16-wk exercise training program in women with PCOS and cross-sectionally in a group of BMI-matched controls.
Main Outcome Measures: Lipolysis was measured in vitro in isolated adipocytes and in vivo by microdialysis of sc abdominal adipose tissue before and during a hyperinsulinemic euglycemic clamp.
Results: In vitro, baseline and maximal ANP- and isoproterenol-induced lipolysis was markedly reduced in PCOS women. Baseline (P < 0.001) and ANP-(P < 0.01) and isoproterenol-(P < 0.001) mediated lipolysis, however, was remarkably increased after training, independent of changes in body weight and sex hormones. These functional improvements were supported by an increased 1) lipolytic sensitivity for ANP (1.3-fold; P < 0.05); 2) lipolytic responsiveness for isoproterenol (1.7-fold; P < 0.01); and 3) postreceptor-acting agent dibutyryl-cAMP (activating cAMP-dependent protein kinase) (2.1-fold; P < 0.05). In vivo, the lipolytic responsiveness to isoproterenol was also reduced in PCOS and tended to increase after exercise training. The insulin suppression of lipolysis during the hyperinsulinemic euglycemic clamp was also reduced in PCOS.
Conclusions: Together, these data show that the regulation of lipolysis by the main endocrine hormones is impaired in women with PCOS. These lipolytic defects can be partly reversed by aerobic exercise training independent of changes in body fat mass and sex hormones.
The regulation of lipolysis by the main endocrine hormones is impaired in women with PCOS, and these lipolytic defects can be partly reversed by aerobic exercise training independent of changes in body fat mass and sex hormones.
Polycystic ovary syndrome (PCOS) is the most common female endocrinopathy, affecting 5–10% of women of reproductive age. PCOS is defined by the presence of clinical or biochemical indicators of androgen excess (e.g. elevated testosterone or hirsutism), menstrual irregularity, and polycystic ovaries (1). A growing body of evidence suggests that PCOS is associated with central obesity, insulin resistance, and hyperinsulinemia (2). Women with PCOS have a substantially higher risk for developing type 2 diabetes and cardiovascular diseases (1, 2).
Adipocytes of women with PCOS are insulin resistant in vitro (3), and multiple defects in catecholamine-mediated lipolysis have been reported in adipocytes of women with PCOS, including reduced levels of β2-adrenoceptors, cAMP-dependent protein kinase (PKA) RIIβ subunit, and hormone-sensitive lipase (HSL) protein content (4, 5, 6). The specificity of the lipolytic resistance to catecholamines observed in women with PCOS is still unclear. Besides catecholamines, insulin and atrial natriuretic peptide (ANP) are vital physiological regulators of human adipocyte lipolysis (7, 8). ANP stimulates lipolysis through a cGMP signaling pathway, activates a cGMP-dependent protein kinase G-Iα, and phosphorylates HSL and perilipin-A (9, 10). It is unknown whether ANP-induced lipolysis is impaired in insulin resistance syndrome such as PCOS.
The lipolytic resistance to catecholamines can be reduced by weight loss but not by oral contraceptive treatment, suggesting that factors other than hyperandrogenicity modulate lipolysis in obese women with PCOS (11). Furthermore impaired β-adrenergic receptor sensitivity can be improved by exercise training in overweight and insulin-resistant obese men in both the basal state and in response to local stimulation by catecholamines (12, 13, 14). We hypothesized therefore that ANP and insulin regulation of lipolysis would be impaired in women with PCOS and that aerobic exercise training would attenuate the functional defects in lipolysis observed in these women independently of weight loss and androgenic state. The aim of the study was to investigate the hormonal regulation of lipolysis, in vitro in isolated adipocytes and in vivo in sc adipose tissue by microdialysis, in women with PCOS vs. body mass index (BMI)-matched controls, and to investigate the ability of aerobic exercise training to override the defects in lipolysis observed in women with PCOS.
Subjects and Methods
Subjects
Eight obese women diagnosed with PCOS and seven obese BMI-matched control women without clinical signs of hyperandrogenicity were recruited in the study (Table 1). The diagnosis of PCOS was confirmed by menstrual irregularity (oligo- or amenorrhea) and either clinical (hirsutism score) or biochemical signs of androgen excess (elevated free testosterone). Other causes of oligomenorrhea (hyperprolactinemia, congenital adrenal hyperplasia, Cushing’s syndrome) were excluded by medical history, and further laboratory tests were ordered in susceptible subjects. Women in the control group were excluded for exercise training, use of contraceptive medications, and menstrual cycle irregularity together with androgen excess. All subjects were nonsmoking and were taking no medication. The protocol was approved by the institutional review board of the Pennington Biomedical Research Center, and all volunteers gave written informed consent.
TABLE 1.
Clinical characteristics of the obese control and PCOS women at baseline and after 16 wk of aerobic training in PCOS women
| Controls at baseline (n = 7) | PCOS (n = 8) | |||||
|---|---|---|---|---|---|---|
| Baseline | Wk 16 | P value | ||||
| Age (yr) | 43 ± 6 | 25 ± 12 | 25 ± 2 | NS | ||
| Weight (kg) | 85.1 ± 4.3 | 84.6 ± 5.8 | 83.3 ± 6.5 | NS | ||
| BMI (kg/m2) | 31.5 ± 0.9 | 32.0 ± 1.6 | 31.8 ± 2.0 | NS | ||
| Waist-to-hip ratio | 0.87 ± 0.02 | 0.85 ± 0.02 | 0.86 ± 0.02 | NS | ||
| Body fat (%) | 40.4 ± 2.3 | 38.2 ± 1.8 | 36.8 ± 2.1 | NS | ||
| Fat mass (kg) | 34.5 ± 2.9 | 32.9 ± 3.8 | 31.5 ± 4.1 | NS | ||
| Fat free mass (kg) | 50.6 ± 2.9 | 51.6 ± 2.3 | 51.8 ± 2.5 | NS | ||
| Fat cell size (μl) | 0.78 ± 0.09 | 0.78 ± 0.10 | 0.82 ± 0.08 | NS | ||
| Fasting FFA (mmol/liter) | 0.70 ± 0.04 | 0.70 ± 0.1 | 0.32 ± 0.03 | <0.001 | ||
| Fasting insulin (μIU/ml) | 15.8 ± 2.5 | 22.2 ± 7.0 | 17.0 ± 3.3 | NS | ||
| GDR (mg · min−1 · kg−1) | 3.7 ± 0.5 | 4.3 ± 0.6 | 5.7 ± 0.7 | <0.001 | ||
| Total testosterone (ng/dl) | 35.4 ± 4.1 | 88.6 ± 13.42 | 86.0 ± 16.9 | NS | ||
| Free androgen index | 5.1 ± 0.5 | 17.5 ± 5.32 | 17.2 ± 5.6 | NS | ||
| VO2max (ml · min−1 · kg−1) | 20.6 ± 1.2 | 27.5 ± 1.31 | 30.9 ± 1.7 | 0.009 | ||
Data are expressed as mean ± sem. The free androgen index was calculated as the ratio of total testosterone divided by SHBG (both expressed in the same units) and multiplied by 100. For conversion to SI units, multiply insulin by 6.945 (pmol/liter) and total testosterone by 0.0347 (nmol/liter). NS, Non significant.
P < 0.05, compared to control.
P < 0.01 compared to control.
Design of the study
All women were examined at baseline, and the women with PCOS were reexamined after 16 wk of aerobic exercise training. Subjects were investigated at 0530 h after an overnight fast and stay in the inpatient of the Pennington Biomedical Research Center clinic. Insulin sensitivity (by euglycemic hyperinsulinemic clamp), body composition (by dual x- ray absorptiometry), aerobic capacity [by maximum oxygen uptake (VO2max) test], in situ adipose tissue lipolysis (by microdialysis), and in vitro lipolysis (in isolated adipocytes) were measured at baseline for both groups and again after 16 wk of aerobic exercise training for the PCOS group. Blood samples and adipose tissue biopsies were also collected.
Exercise training
Aerobic exercise was performed under supervision at the Pennington Health and Fitness Center five times per week. Aerobic exercise was prescribed on an individual basis with the objective to achieve specified exercise energy expenditure (ExEE) in each session. During the first 4 wk, the target ExEE was 4% of the participants’ estimated energy requirement for weight maintenance, and it was incremented to 6% for wk 5–8, to 8% for wk 9–12, and to 10% for wk 13–16. The energy requirement was calculated with a previously published equation of energy intake for weight maintenance established in free-living participants at the Pennington Center: energy requirement (kilocalories per day) = 1625 + 31.8 (fat free mass in kilograms) + 1.5 (fat mass in kilograms) − 187 (for females) (15). All exercises were performed on a treadmill at 55% VO2max, a moderate intensity. The necessary speed and gradient to achieve the ExEE was estimated from a linear regression of O2 uptake and workload during the VO2max test, and the ExEE was confirmed once during each 4-wk interval by indirect calorimetry. Heart rate was monitored during all sessions to verify ExEE. The exercise time necessary to complete the energy expenditure target was 23 ± 1 min per session during wk 1–4, 35 ± 1 min per session during wk 5–8, 47 ± 2 min per session during wk 9–12, and 58 ± 2 min per session during wk 13–16.
Hyperinsulinemic euglycemic clamp
Insulin sensitivity was measured by a euglycemic hyperinsulinemic clamp as previously described (16). Forty-eight hours before presenting to the Pennington inpatient clinic, participants consumed a weight-maintaining diet consisting of 35% fat, 15% protein, and 50% carbohydrate (prepared by the metabolic kitchen) and were asked to refrain from vigorous physical activity.
At 0530 h, after an overnight fast, an iv catheter was placed in an antecubital vein for infusion of insulin and 20% glucose during the clamp. A second catheter was placed retrograde in a dorsal vein of the contralateral hand for blood withdrawal. A primed infusion of regular insulin (80 mU/min · m2) was initiated and continued for approximately 120 min. Plasma glucose was maintained at 90 mg/dl in all participants. Arterialized plasma glucose was measured at 5-min intervals, and exogenous glucose (20% solution) was infused at a variable rate to maintain plasma glucose concentration at approximately 90 mg/dl. Glucose and insulin were measured in four independent blood plasma samples 10 min apart at baseline and again at steady-state after approximately 2 h. Glucose disposal rate (GDR) was adjusted for kilograms of body weight.
Adipose tissue biopsy
After the insertion and calibration of the microdialysis probe, a topical anesthesia was injected, and a 1.0-cm incision was made approximately 5 cm to the umbilicus (on the contralateral side to the microdialysis probe). A needle biopsy was performed to collect approximately 1 g of sc adipose tissue.
Studies of in vitro lipolysis
Subcutaneous adipose tissue samples (400–600 mg) were cut with scissors into small pieces under aseptic conditions. Adipocytes were isolated by collagenase digestion according to Rodbell (17). After digestion, the suspension was filtered (210-μm filter) and washed three times with PBS, and adipocytes were brought to a suitable dilution (2000–3000 cells/100 μl) into Krebs Ringer bicarbonate buffer containing glucose (5.55 mmol/liter) and 20 mg/ml of BSA at pH 7.4. Isolated adipocytes were then incubated with 5 μl of pharmacological agents in a final volume of 100 μl during 90 min at 37C under gentle shaking at 120 cycles/min. After incubation, 30 μl of medium in duplicate was taken to measure glycerol (used as an index of lipolysis) using the free glycerol reagent (Sigma-Aldrich, St. Louis, MO), and total lipids were extracted gravimetrically (18). Lipolysis was expressed as micromoles of glycerol per 100 mg of lipids as previously described (10, 19).
Studies of in vivo lipolysis
A microdialysis catheter (CMA 60; CMA Microdialysis, Stockholm, Sweden) with a 30-mm long membrane was inserted into the abdominal sc abdominal tissue under local anesthesia. The catheter was connected to a microdialysis pump (CMA 107 Microdialysis Pump) and continuously perfused with sterile Ringers solution (139 mmol/liter sodium, 2.7 mmol/liter potassium, 0.9 mmol/liter calcium, 140.5 mmol/liter chloride, 2.4 mmol/liter bicarbonate, 5.6 mmol/liter glucose) supplemented with ethanol (1.7 g/liter) to estimate changes in adipose tissue blood flow (ATBF) (20, 21).
The probe and tubing are first flushed for 10 min at 10 μl/min, and the accumulated dialysate is discarded. The perfusion system is then calibrated. To begin, the flow rate is set to 2.0 μl/min for 30 min, and the dialysate accumulated is discarded. Next, the flow rate is reduced to 0.5 μl/min, and a single dialysate fraction is collected at 30 min. The flow rate is increased to 2.0 μl/min, and three 10-min dialysate fractions are collected. These samples are used to determine baseline in vivo lipolysis. To assess the in vivo effects of a β-adrenergic agonist on lipolysis, 1 μmol/liter isoproterenol hydrochloride (Isuprel; Hospira, Inc., Lake Forest, IL) in ringer solution was perfused for 30 min. After these baseline studies were complete, the hyperinsulinemic euglycemic clamp was initiated, during which the probe was perfused with Ringer solution only. Dialysate fractions continued to be collected every 10 min throughout the duration of the clamp. A comprehensive scheme of the experimental protocol is provided in Fig. 3A.
Fig. 3.
A, Scheme of the microdialysis protocol before and during the hyperinsulinemic euglycemic clamp. B, Time-course of sc adipose tissue lipid mobilization (extracellular glycerol in μmol/liter) in obese control and PCOS women at baseline. The −10 and 0 time points represent the baseline glycerol measurement. Isoproterenol (1 μmol/liter) was infused for 30 min as indicated by the black bar on the x-axis. The euglycemic hyperinsulinemic clamp was performed from 30 to 150 min. C, AUC calculated during the 30-min lipolytic response to isoproterenol in obese controls and PCOS women at baseline. D, AUC of extracellular glycerol calculated during the 120-min euglycemic hyperinsulinemic clamp in obese controls and PCOS women at baseline. *, P < 0.05 compared with control.
The microvials were stored in specialized microvial racks at −80 C until analyzed. Dialysate concentrations of glycerol were measured in duplicate by an automated spectrophotometric kinetic enzymatic analyzer (CMA 600; CMA Microdialysis, Solna, Sweden), and ethanol by colorimetric assay as previously described (20, 22).
Gene expression by real-time quantitative PCR
Total RNA from adipose tissue biopsies was isolated with Trizol reagent (Invitrogen, Carlsbad, CA). The quantity and integrity of the RNA was confirmed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All primers and probes were designed using Primer Express version 2.1 (Applied Biosystems-Roche, Branchburg, NJ). Sequences of primers and probes are available in Supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Real-time quantitative RT-PCRs were performed as one-step reactions in ABI PRISM 7900 (Applied Biosystems) using the following parameters: one cycle of 48 C for 30 min, then 95 C for 10 min, followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min. For all assays performed using Taqman primers and probe, the ribosomal phosphoprotein large P0 gene was used as internal control. The relative standard curve method was used to calculate the quantity of each gene. All expression data were normalized by dividing the quantity of the target gene by the quantity of the internal control.
Hormone and metabolite determination
Fasting free fatty acid (FFA) concentrations were determined in serum by enzymatic assay (Wako Chemicals USA, Inc., Richmond, VA). Insulin was determined in serum by immunoassay (Linco Research Inc., St. Charles, MO). Serum testosterone and SHBG were both measured by an automated chemiluminescent immunoassay on the Immulite 2000 (Siemens Healthcare Diagnostics, Deerfield, IL).
Statistical analyses
All statistical analyses were performed using GraphPad Prism 4.0 for Windows (GraphPad Software, Inc., San Diego, CA). Two-tailed paired and unpaired t tests were performed to determine the effect of exercise training and PCOS on glycerol release (dependent variable), respectively. Two-way ANOVA (including drug concentration (isoproterenol or ANP) and group (control vs. PCOS), or drug concentration and exercise training (before vs. after) as independent variables] were performed. Bonferroni’s post hoc multiple comparison tests were performed when a significant interaction term was observed between the two independent variables. Spearman correlations were performed to determine the relationship between changes in lipolysis and clinical variables. All values in figures and tables are presented as mean ± sem. Statistical significance was set at P < 0.05.
Results
Clinical characteristics of the subjects
The obese PCOS women showed a hyperandrogenic state despite similar indices of body composition compared with the obese control women (Table 1). The control women were slightly older compared with the PCOS women and had similar whole-body insulin sensitivity.
Effect of 16 wk of aerobic exercise training
After 16 wk of exercise training, there was no change in body weight, fat mass, or fat-free mass (Table 1). Insulin sensitivity (GDR) increased by 35 ± 6%, and fasting insulin concentrations decreased by 24%, whereas aerobic fitness increased as expected (12 ± 1%; P < 0.05). There was no significant change in serum total testosterone (−5 ± 13%) or SHBG (3 ± 9%).
Comparison of in vitro lipolysis between women with PCOS and BMI-matched controls
Basal lipolysis
In comparison to BMI-matched control women, baseline lipolysis was reduced in women with PCOS despite similar average adipocyte size (Table 2) and negatively correlated with the percentage of body fat (r = −0.90; P < 0.01).
TABLE 2.
Lipolytic sensitivity and responsiveness of catecholamines and ANP in adipocytes of obese control vs. PCOS women at baseline and after 16 wk of aerobic exercise training in PCOS women
| Controls at baseline (n = 7) | PCOS (n = 8) | |||||
|---|---|---|---|---|---|---|
| Baseline | Wk 16 | P value | ||||
| Basal lipolysis | 0.32 ± 0.04 | 0.11 ± 0.012 | 0.34 ± 0.04 | 0.0001 | ||
| Isoproterenol | ||||||
| EC50 (nmol/liter) | 15.6 ± 5.3 | 148.5 ± 65.41 | 78.9 ± 30.0 | NS | ||
| pD2 | 8.00 ± 0.18 | 7.22 ± 0.291 | 7.35 ± 0.20 | NS | ||
| Maximum | 0.50 ± 0.06 | 0.32 ± 0.051 | 0.55 ± 0.07 | 0.003 | ||
| ANP | ||||||
| EC50 (nmol/liter) | 1.08 ± 0.48 | 4.61 ± 1.201 | 3.33 ± 1.49 | NS | ||
| pD2 | 9.54 ± 0.39 | 8.44 ± 0.121 | 9.14 ± 0.29 | 0.03 | ||
| Maximum | 0.45 ± 0.10 | 0.32 ± 0.05 | 0.37 ± 0.07 | NS | ||
| Postreceptor signaling agents | ||||||
| Dibutyryl-cAMP (5 mmol/liter) | 0.21 ± 0.06 | 0.28 ± 0.04 | 0.58 ± 0.10 | 0.02 | ||
| 8-Bromo-cGMP (5 mmol/liter) | 0.32 ± 0.06 | 0.30 ± 0.05 | 0.40 ± 0.12 | NS | ||
Data are expressed as mean ± sem of changes in glycerol release over baseline (μmol/100 mg lipids). pD2 represents −log[EC50], an index of lipolytic potency. NS, Non significant.
P < 0.05, compared to control.
P < 0.001 compared to control.
Hormone-stimulated
The lipolytic response to isoproterenol, however, was markedly reduced in adipocytes of women with PCOS as previously described (4, 6). Isoproterenol maximal-induced lipolysis was 0.43 ± 0.05 and 0.82 ± 0.12 μmol/100 mg of lipids for PCOS vs. control, respectively (P < 0.01). Both the lipolytic responsiveness to isoproterenol (maximal-induced lipolysis) (1.6-fold; P < 0.05) and the lipolytic sensitivity (9-fold; P < 0.05) were reduced in adipocytes of PCOS women (Fig. 1A and Table 2). Maximal-stimulated isoproterenol lipolysis was negatively related to serum testosterone levels (r = −0.67; P < 0.05) and the free androgen index (r = −0.88; P < 0.01).
Fig. 1.
Dose-response lipolytic effect of isoproterenol (β-agonist) (A) and ANP (B) in human sc adipocytes in women with PCOS vs. matched controls. P indicates a significant effect of PCOS by ANOVA. A significant interaction between group (PCOS or control) and isoproterenol concentration was observed (P = 0.0024). **, P < 0.01 compared with control at the respective concentration.
A new finding is that the lipolytic sensitivity of ANP was reduced (4-fold; P < 0.05) in adipocytes of PCOS women (Fig. 1B and Table 2). ANP maximal-induced lipolysis was 0.44 ± 0.06 and 0.81 ± 0.14 μmol/100 mg of lipids in PCOS vs. control, respectively (P < 0.05). ANP lipolytic responsiveness was not affected and was negatively correlated with fat cell size (r = −0.88; P < 0.01). Interestingly, the lipolytic potency (pD2) of ANP was negatively related to the percentage of body fat (r = −0.93; P < 0.01). To further characterize the postreceptor mechanism, isolated adipocytes were incubated with two postreceptor signaling molecules, dibutyryl-cAMP to activate PKA and 8-bromo-cGMP to activate cGMP-dependent protein kinase (PKG). There was no difference in the lipolytic responsiveness to dibutyryl-cAMP and 8-bromo-cGMP between the two groups (Table 2). In summary, both ANP- and catecholamine-induced lipolysis was impaired in adipocytes of PCOS women, apparently due to defects at the receptor level.
Effect of exercise training on in vitro lipolysis in obese women with PCOS
Basal lipolysis
Baseline lipolysis increased after exercise training and was restored to the level of baseline adipocyte lipolysis observed in control obese women (Table 2). This occurred in the absence of changes in fat cell size and body composition (Table 1), and no change in mRNA levels of the main lipases HSL, adipose triglyceride lipase (ATGL), and its coactivator comparative gene identification of 58 kDa were found before and after training (Supplemental Table 2).
Hormone-stimulated
Catecholamine lipolytic responsiveness was also fully restored in PCOS women after exercise training (Fig. 2A and Table 2). This was mainly due to improvement in postreceptor cAMP signaling (0.39 ± 0.03 and 0.91 ± 0.08 μmol/100 mg of lipids for before and after training, respectively; P < 0.001) (Table 2). Interestingly, the lipolytic sensitivity to ANP was improved independently of changes in postreceptor cGMP signaling effects on lipolysis, possibly suggesting an adaptation at the receptor level (0.41 ± 0.04 and 0.63 ± 0.09 μmol/100 mg of lipids for before and after training, respectively) (Fig. 2B and Table 2). We did not observe any difference in natriuretic peptide receptor A gene expression between baseline and wk 16 of exercise training (Supplemental Table 2). Of interest, there was a positive relationship between the changes in dibutyryl-cAMP (r = 0.64; P < 0.05) and 8-bromo-cGMP (r = 0.71; P < 0.05)-stimulated lipolysis and the change in aerobic capacity (VO2max).
Fig. 2.
Dose-response lipolytic effect of isoproterenol (β-agonist) (A) and ANP (B) in human sc adipocytes in women with PCOS before and after 16 wk of aerobic exercise training. P indicates a significant effect of exercise training by ANOVA.
Comparison of in vivo lipid mobilization between women with PCOS and BMI-matched controls
Baseline
Average baseline extracellular glycerol concentration was lower in women with PCOS compared with controls (54.4 ± 5.7 vs. 91.6 ± 16.8; P < 0.05) (Fig. 3B).
Catecholamine-stimulated
Isoproterenol-induced lipid mobilization measured by microdialysis of abdominal sc adipose tissue was also reduced in women with PCOS (Fig. 3B). The overall lipolytic responsiveness of isoproterenol was blunted in women with PCOS as indicated by a 58% (P < 0.05) reduction in the area under the curve (AUC) calculated during isoproterenol infusion (Fig. 3C).
Insulin suppression of lipolysis
Interestingly, the suppression of lipid mobilization by insulin (measured by the AUC of extracellular glycerol during the 120-min of hyperinsulinemic euglycemic clamp) was also reduced by 48.8% (P < 0.05) in women with PCOS (Fig. 3D). ATBF, assessed by the ethanol outflow-to-inflow ratio, tended to increase in response to isoproterenol infusion and remained unaffected during the clamp as previously shown (data not shown) (14).
Effect of exercise training on in vivo lipid mobilization in obese women with PCOS
Baseline
The average baseline extracellular glycerol concentration did not increase significantly after exercise training in PCOS women [54.4 ± 5.7 vs. 67.9 ± 14.8 μmol/liter; nonsignificant (NS)] (Fig. 4A).
Fig. 4.
A, Time-course of sc adipose tissue lipid mobilization (extracellular glycerol in μmol/liter) in women with PCOS before and after 16 wk of aerobic exercise training. The −10 and 0 time points represent the baseline glycerol measurement. Isoproterenol (1 μmol/liter) was infused for 30 min as indicated by the black bar on the x-axis. The euglycemic hyperinsulinemic clamp was performed from 30 to 150 min. B, AUC calculated during the 30-min lipolytic response to isoproterenol in PCOS women before and after 16 wk of aerobic exercise training. C, AUC of extracellular glycerol calculated during the 120-min euglycemic hyperinsulinemic clamp in women with PCOS before and after 16 wk of aerobic exercise training. *, P < 0.05 compared with before training.
Catecholamine-stimulated
Interestingly, isoproterenol-induced lipid mobilization was significantly increased at 10 min after infusion and overall tended to be higher after exercise training as shown by the AUC calculated during isoproterenol infusion (Fig. 4B).
Insulin-suppression of lipolysis
There was no significant improvement in the suppression of lipid mobilization (AUC extracellular glycerol) during the clamp after exercise training (Fig. 4C). No significant changes in ATBF at baseline and during the clamp were observed after exercise training (data not shown).
Discussion
The main finding of the present study is that adipocyte defects in lipolysis observed in PCOS are not restricted to the lipolytic resistance to catecholamines. The stimulation of lipolysis by ANP and the suppression of lipolysis by insulin, two vital regulators of lipolysis in human adipocytes, are also blunted in obese women with PCOS. Interestingly, these multiple functional defects in lipolysis can be partly recovered after 16 wk of aerobic exercise training independently of changes in body fat mass and systemic androgen status.
In the present study, we investigated the regulation of lipolysis by ANP, catecholamines, and insulin in vitro and for the first time in vivo in women with PCOS compared with BMI-matched control. The PCOS women had similar fasting insulin and whole-body insulin sensitivity compared with the obese control women. Interestingly, baseline lipolysis was reduced both in isolated adipocyte and in vivo in sc abdominal adipose tissue in women with PCOS. This is likely to be the consequence of reduced HSL protein expression because ATGL protein expression is not affected in PCOS (23). Thus, the insulin suppression of lipolysis in vivo measured during the hyperinsulinemic euglycemic clamp was remarkably reduced (49%) in women with PCOS, suggesting that adipocytes of PCOS women are more insulin resistant. This feature is consistent with previous findings in vitro (3, 24), and it suggests that adipose tissue insulin resistance could develop at an early stage in women with PCOS. It could be speculated that adipose tissue insulin resistance could contribute to chronic elevation of plasma FFA and impact other insulin-sensitive tissues such as skeletal muscle and liver. This is somehow consistent with the fact that older PCOS women are more insulin resistant compared with BMI-matched controls and have a higher risk of developing type 2 diabetes (1, 2, 24). Additionally, we show that adipose tissue insulin sensitivity did not improve significantly after 16 wk of aerobic exercise training despite a clear increase in whole-body glucose disposal. Thus, neither adipocyte size nor total body fat mass was changed after training, suggesting that adipocyte insulin sensitivity might be primarily dependent on adipocyte size and/or other unknown mechanisms.
Multiple defects in catecholamine-induced lipolysis in PCOS have been previously reported by Arner and collaborators (5, 6, 23), including reduced protein expression of HSL, β2-adrenoceptors, and the PKA RIIβ subunit. This study shows that besides catecholamine, others major endocrine pathways regulating lipolysis are affected in PCOS. We show specifically that ANP-mediated lipolysis and insulin-suppressed lipolysis are markedly reduced in women with PCOS. ANP is produced by the left atria of the heart and plays a major role in the regulation of sodium water balance in mammals and humans (25, 26). Recently, ANP was identified as a powerful lipolytic peptide in vitro in isolated adipocytes (27) and in vivo in humans (7, 28). It was shown that ANP induces HSL and perilipin-A phosphorylation through a cGMP/PKG pathway (9). It is still unclear whether ANP-induced lipolysis is impaired with obesity and insulin resistance. In this study, we show that the lipolytic sensitivity of ANP was briskly reduced in adipocytes of women with PCOS. This defect could occur at the receptor level because the lipolytic effect of 8-bromo-cGMP, activating downstream PKG, was preserved. We were not able to detect any significant variation in the expression level of the ANP receptor natriuretic peptide receptor A. We also confirmed the lipolytic resistance to catecholamines in adipocytes of women with PCOS. Both the lipolytic sensitivity and responsiveness of isoproterenol were markedly reduced in adipocytes of PCOS. The lipolytic responsiveness of isoproterenol was negatively correlated to the serum free testosterone levels in adipocytes of women with PCOS (r = −0.67; P < 0.05). This is consistent with a recent report showing that testosterone directly inhibits β2-adrenoceptors expression in cultured primary human preadipocytes (29). Thus, we investigated for the first time isoproterenol-induced lipid mobilization in vivo by microdialysis of sc abdominal adipose tissue, the overall lipolytic responsiveness of isoproterenol in sc abdominal adipose tissue was reduced by 58% in women with PCOS.
We next investigated whether aerobic exercise training was sufficient to overcome the multiple functional defects in lipolysis of women with PCOS. Baseline lipolysis measured both in vitro and in situ was significantly increased after exercise training and restored to the level of the control group. This effect occurred independently of changes in body composition, fat cell size, and changes in gene expression of the main adipocyte lipases HSL and ATGL. However, we cannot rule out changes in adipocyte HSL or ATGL protein expression and/or enzyme activity after exercise training. Indeed, other studies have reported an elevated HSL protein content and activity in adipose tissue of exercise-trained rat compared with sedentary control littermates (30, 31). Of interest, both ANP lipolytic sensitivity and isoproterenol lipolytic responsiveness were partly recovered after exercise training in women with PCOS. This is likely due to a combination of receptor and postreceptor signaling molecular changes leading to facilitated PKA- and PKG-stimulated HSL phosphorylation and activity. This is supported by improved lipolytic response of the postreceptor signaling agent’s dibutyryl-cAMP and 8-bromo-cGMP in adipocytes of women with PCOS after exercise training. Thus, the lipolytic response to these compounds positively correlated with the improvement of aerobic capacity, suggesting that exercise training may induce beneficial effects on adipocyte lipolysis without significant changes in body fat mass or adipocyte size. The underlying mechanisms should be investigated. We next investigated isoproterenol-stimulated lipolysis in situ in abdominal adipose tissue of women with PCOS after exercise training. We found a nonsignificant trend for increased lipid mobilization in response to isoproterenol after exercise training. Previous studies in overweight and obese males have shown beneficial effects of exercise training on catecholamine-induced lipolysis (12, 13, 14). In the present study, the higher lipid mobilization could have been masked by an increased ATBF that increases glycerol washout from the tissue and reduces extracellular glycerol concentrations. We used the nonquantitative ethanol outflow-to-inflow technique to estimate ATBF as previously described (13, 20). However, this technique does not permit quantification of changes in ATBF in response to a physiological intervention such as exercise training (32, 33).
In conclusion, this study shows that lipolytic defects in women with PCOS are not restricted to catecholamine resistance. We show that ANP and insulin regulation of lipolysis is also blunted in adipocytes of PCOS. These functional defects can be partly recovered after a 16-wk aerobic exercise training program independently of changes in body fat mass and androgenic state. Future studies should investigate the underlying mechanisms. This study also highlights the possibility that exercise prescription combined with a low-calorie diet may be of importance in women with PCOS to enhance lipolysis and weight loss.
Acknowledgments
We thank Professors Eric Ravussin and Steven Smith for pivotal advice in the development of the experimental design and critique of the manuscript, and Stacy Carling for excellent technical assistance. We also thank Mandy Shipp, Laura Daray, and the nursing staff of the Pennington Biomedical Research Center Inpatient Unit. Special thanks goes to the study participants, without whom these questions could not have been answered.
Footnotes
Disclosure Summary: The authors have nothing to disclose.
First Published Online April 14, 2009
Abbreviations: ANP, Atrial natriuretic peptide; ATBF, adipose tissue blood flow; ATGL, adipose triglyceride lipase; AUC, area under the curve; BMI, body mass index; ExEE, exercise energy expenditure; FFA, free fatty acid; GDR, glucose disposal rate; HSL, hormone-sensitive lipase; PCOS, polycystic ovary syndrome; pD2, lipolytic potency; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; VO2max, maximum oxygen uptake.
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