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Original Studies |
Department of Medicine, Research Center, and Department of Gynecology and Obstetrics (I.E., K.C., A.B.), Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden
Address all correspondence and requests for reprints to: Hans Wahrenberg, M.D., Center of Metabolism and Endocrinology, Department of Medicine M63, Huddinge Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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WR caused a 50% reduction of basal lipolysis rate and a 5- to 7-fold
increased noradrenaline and terbutaline sensitivity
(P < 0.02); the latter could be ascribed to a
2-fold increased ß2-adrenoceptor density
(P < 0.02) as determined with radioligand binding.
There was no change with regard to dobutamine
(ß1-adrenoceptor sensitivity) or clonidine,
(
2-adrenoceptor sensitivity) or to
ß1-adrenoceptor density. OC treatment did not influence
the basal lipolysis rate or ß2- or
2-adrenoceptor sensitivity, but lowered the
ß1-adrenoceptor sensitivity 7-fold (P
< 0.03) without a reduction in ß1-adrenoceptor density.
The OC treatment effect was not observed when forskolin and dibutyryl
cAMP, acting on adenylate cyclase or protein kinase A, respectively,
were used, suggesting a partial uncoupling of
ß1-adrenoceptors. WR therapy, but not OC therapy, caused,
in addition to changes in lipolysis function, improved in
vivo insulin sensitivity and lower plasma noradrenaline levels.
These findings suggest that factors other than hyperandrogenicity
modulate lipolysis regulation in obese subjects with PCOS. Disturbances
in sympathetic pathways could be of pathogenic importance.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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2-ARs. Subcutaneous adipose tissue is the major fat
depot, constituting about 80% of all fat. In the visceral fat depot, a
third ß-AR, namely ß3, has been found to be of
additional importance for lipolysis regulation (7). Lipolytic
noradrenaline resistance due to reduced ß2-AR density has
been found in sc fat cells of upper body obese women as well as in
young nonobese women with PCOS (8, 9). In the latter group there was an
additional defect in the hormone-sensitive lipase complex, which is the
final rate-limiting step in lipolysis regulation. Thus, both elevated
androgens and impaired lipolysis due to catecholamine resistance could
be primary factors of a vicious circle leading to abdominal obesity
aggravating insulin resistance. The present study was undertaken to determine whether lipolysis regulation could be influenced in obese women with PCOS by two different intervention programs with the aim of reducing the fT level by weight reduction or by oral anticonception therapy to gain more knowledge about how these factors interact and are regulated in women with the PCOS. Subcutaneous fat cells were incubated in vitro with agents acting on lipolysis at different steps in lipolysis regulation from adrenoceptors to the hormone-sensitive lipase complex. In addition, the quantities of ß1- and ß2-ARs were determined by radioligand binding techniques in the adipocytes.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Twenty obese women with the diagnosis of PCOS were recruited among patients referred to the Department of Obstetrics and Gynecology. The diagnosis was based on clinical findings, such as infertility, hirsutism, oligomenorrhea, a ratio of serum total T to SHBG greater than 0.063, and a LH/FSH ratio above 1 on a minimum of two occasions during a period of 6 months before examination. Polycystic ovaries were confirmed by gynecological examination and transvaginal ultrasound technique. Obesity was defined as a body mass index above 27 kg/m2. No subject was taking any medication. The women were openly offered participation in different intervention programs, either a weight reduction (WR) program with a very low calorie diet (VLCD), described previously (10), or a 3-month treatment program with a low dose combined oral contraceptive (OC) pill (Orthonett Novum, Janssen-Cilag, Madrid, Spain) containing 0.5 mg norethisterone and 35 µg ethinyl estradiol. The women were informed about these 2 different methods to reduce the androgen level to improve female sex functions. Ten women chose the weight reduction program, and the remaining 10 women volunteered for the OC treatment. The women were examined at 0800 h after an overnight fast. The waist/hip ratio, body mass index, and blood pressure were determined. After 30 min of rest in the supine position, a serum sample was obtained for analysis of hormones, SHBG, and glucose. Serum concentrations of cortisol, T, SHBG, glucose, cholesterol, and triglycerides were determined at the hospital’s clinical chemistry laboratory by established routine methods. Apparent concentrations of fT were calculated from values of total T, SHBG and a fixed albumin concentration of 40 g/L by successive approximation using a computer program based upon equation system derived from the law of mass action (11, 12). Serum insulin was measured by RIA using a commercial kit obtained from Pharmacia & Upjohn, Inc. (Stockholm, Sweden). A sc fat biopsy of adipose tissue about 3 g was obtained during local anesthesia from the abdominal region randomly from the left or right side at the middle to the umbilicus (13). Seventeen of the 20 women with PCOS adhered to the protocol and were reexamined after a 3-month period of OC therapy (n = 8) or VLCD treatment (n = 9). The women in the WR group underwent the second examination 2–4 weeks after finishing the VLCD to avoid acute catabolic effects of the diet. During the examination period they were given an isocaloric diet, which kept their weight stable (mean difference, -0.34 kg between 0–4 weeks after finishing the VLCD). The second examination was performed in the same way as the first one, except the biopsy was taken from the contralateral side.
The study was approved by the committee on ethics at the Karolinska Institute. The study protocol was explained in detail to each participant, and her consent was obtained.
Isolation of fat cells and determination of fat cell size and number
Isolated fat cells were prepared according to the collagenase method described by Rodbell (14). Direct microscopic determination of the diameter of 100 fat cells was performed as described previously (15). The coefficient of variation was about 3%. Mean fat cell volume, surface area, and weight were determined as previously described (16). The number of fat cells incubated was calculated as follows. The lipid content of the aliquot was determined gravimetrically after solvent extraction. The lipid content of the incubated fat cells was then divided with the mean fat cell weight, assuming that the lipids constitute more than 95% of the fat cell weight.
Lipolysis experiments
Isolated fat cells were incubated as described in detail
previously (17). In brief, about 5,000–10,000 cells/mL were incubated
in duplicate at 37 C in Krebs-Ringer phosphate (pH 7.4) containing
albumin (20 g/L), glucose (1 g/L), and ascorbic acid (0.1 g/L) in the
absence or presence of increasing concentrations
(10-16–10-4 mol/L) of noradrenaline
(endogenous agonist stimulating ß- and 2-ARs),
isoprenaline (nonselective ß-AR agonist), terbutaline (selective
ß2-AR agonist), dobutamine (selective ß1-AR
agonist), clonidine (selective 2-AR agonist), forskolin
(adenylyl cyclase stimulator), and dibutyryl cAMP
(phosphodiesterase-resistant cAMP analog). In the clonidine experiments
adenosine deaminase (1 mU/mL) was added to the incubation medium to
remove traces of adenosine, which may interfere with the antilipolytic
effect of clonidine (17). However, the presence of adenosine
deaminase in the incubation medium increased the basal lipolysis
rate by 25–30% and did not influence the results with stimulated
lipolysis. After 2 h, an aliquot was removed for determination of
glycerol. The concentration of agonist causing 25% (EC25),
50% (EC50), 75% (EC75), and 100%
(EC100) of the maximum effect was determined using logistic
sigmoid curve fitting of each individual’s dose-response data by a
computer program (Ultrafit, Biosoft, Ferguson, MO). The
negative logarithm of the EC50 value (pD2) was
defined as AR sensitivity. Mean dose-response curves were constructed
for presentation graphs from the above means (EC25–100) by
a logistic sigmoid curve-fitting technique. The maximum rate of
lipolysis (glycerol release per 107 fat cells) was also
determined as the rate at the maximum effective concentration of the
lipolytic agent.
ß-AR binding studies
Receptor binding studies have been described in detail previously (17). Isolated fat cells 20,000 cells/mL were incubated at 37 C in 0.5 mL Krebs-Ringer phosphate buffer (pH 7.4) containing albumin (5 g/L), glucose (1 g/L), and ascorbic acid (0.1 g/L). Saturation experiments were performed to determine the total amount of ß-ARs. The cells were incubated in duplicate for 60 min with six different concentrations of [125I]cyanopindolol ([125I]CYP). Nonspecific binding determined in the presence of 0.1 µmol/L propranolol was about 30% at low and about 45% at high radioligand concentrations. In duplicate competition experiments performed to determine the fraction of ß2-ARs of the total ß-receptor population, 100 pmol/L [125I]CYP competed with 12 increasing concentrations of the ß2-specific antagonist ICI 118,551 (10-11–10-4 mol/L). Nonspecific binding at 10-4 mol/L ICI 118,551 was about 30% and did not differ from nonspecific binding determined by 0.1 µmol/L propranolol. The binding experiments were evaluated by computerized curve fitting (Kell, Biosoft) (18). The software calculates estimates of maximum total binding capacity (Bmax) obtained from the saturation binding experiments as well as the affinity constants (Kd) and the proportion of ß1- and ß2-ARs accessed from the displacing experiments by ICI 118,551. At the concentrations of [125I]CYP used in these experiments, there is no significant binding to ß3-ARs. Instead, the radioligand binds with homogeneity of ß1- and ß2-ARs, yielding linear Scatchard curves with slopes near 1.
Drug and chemicals
BSA (fraction V, 63F 0748), Clostridium histolyticum collagenase type 1, glycerol kinase from Escherichia coli (G4 509), forskolin, dibutyryl cAMP and D,[SCAP]L-propranolol were obtained from Sigma Chemical Co. (St. Louis, MO). (-)-Isoprenaline hydrochloride was purchased from Hässle (Molndal, Sweden). Terbutaline sulfate was obtained from Draco (Lund, Sweden). Dobutamine hydrochloride was purchased from Eli Lilly & Co. (Indianapolis, IN). Clonidine was obtained from Boehringer Ingelheim GmbH (Rhein, Germany), and ICI 118,551 was purchased from Cambridge Research Biochemist Ltd. (Sessyr, UK). Adenosine deaminase was of calf intestine origin and was supplied by Boehringer Mannheim (Mannheim, Germany), and ATP monitoring regent containing firefly luciferase was purchased from LKB-Wallac, Inc. (Turku, Finland). [125I]CYP was obtained from New England Nuclear Corp. (Boston, MA). All other chemicals were of the highest grade of purity commercial available.
Statistics
Student’s two-tailed t test was used for
comparison of data between (unpaired) and within (paired) groups. The
SD was used as a measure of dispersion of clinical
characteristics data (Table 1
) and the
SEM was used for experimental data when normally
distributed. All statistics were analyzed by means of the Systat
software statistical package (SPSS, Inc., Chicago, IL).
Values for nonnormally distributed experimental parameters, such as
Kd and EC50, were transformed into their
logarithmic form before statistical analysis with parametric tests was
performed. Nonnormally distributed clinical data were expressed as the
median and range, and appropriate nonparametric tests were used for
statistical comparisons.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The VLCD caused a mean weight reduction of 8 ± 3 kg, whereas
weight was unchanged in the OC group. The SHBG level rose significantly
in both groups, but more markedly in the OC group, whereas total serum
T decreased equally in both groups, resulting in a normalized fT level.
There were indirect signs of improved insulin sensitivity, such as
significant lower levels of fasting insulin and blood glucose in the WR
group, but not in the OC group, indicating that insulin resistance was
still present in the latter group. Moreover, plasma levels of
catecholamines and arterial blood pressure fell significantly in the WR
group (but not in the OC group), also indicating a reduced sympathetic
activity (Table 1
). The WR group was about 10 yr older than the OC
group (P < 0.01). There were, however, no other
clinical differences between the groups at baseline before the
intervention.
Lipolysis experiments
Concentration-response curves for agonist-induced lipolysis are
depicted in Fig. 1, and Table 2 displays all relevant lipolysis data
for the two groups. There were no difference at baseline in the basal
rate of lipolysis or the lipolytic dose-response curves of any agonist
between the two groups. The lipolytic sensitivity (PD2) of
the endogenous catecholamine noradrenaline was increased 10-fold after
weight reduction (P < 0.03), whereas treatment with OC
caused an opposite 7-fold decreased sensitivity (P <
0.04). Likewise, the lipolytic sensitivity to the nonselective ß-AR
agonist isoprenaline increased 100-fold after WR (P <
0.03), but decreased 70-fold after OC (P < 0.05).
Analysis of the effect of weight reduction on subtype-selective
agonists showed a significant 8-fold increase in ß2-AR
sensitivity (terbutaline; P < 0.02), whereas the
ß1-AR sensitivity (dobutamine; P = NS)
and 2-AR (clonidine; P = NS)
sensitivities remained unchanged. However, in the group receiving OC
therapy, a 10-fold lower ß1-AR sensitivity
(P < 0.03) was observed, whereas the
ß2-AR and 2-AR sensitivities were
unchanged. Although there were numerically lower means of maximum
lipolytic responsiveness to adrenergic agonists in both groups after
intervention, these differences were far from statistically significant
(P = 0.35–0.75), because of divergent responses among
individuals in both groups. When lipolysis was stimulated at the
adenylyl cyclase level with forskolin or at the level of the protein
kinase complex with the phosphodiesterase-resistant cAMP analog
dibutyryl cAMP, no significant change in the maximum lipolytic response
to these agents was observed in either group. There was a significant
change in adipocyte size in the VLCD group (894 ± 57 pL before
vs. 706 ± 57 after weight reduction; P
< 0.02), whereas in the OC group no significant change occurred
(770 ± 50 pL before and 690 ± 68 pL after therapy). Thus,
the lipolysis rate was expressed per cell number, and ligand binding
data were expressed per cell surface area.
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Weight reduction caused a significant increase in the total
ß-AR density from about 3 to 4 attomoles
(amol)/mm2 (P < 0.05) as determined from
Scatchard analysis of individual saturation experiments with
[125I]CYP (Table 3).
Displacement of [125I]CYP by the selective
ß2-antagonist ICI 118,551 showed shallow biphasic curves
significantly better fitted to a two-site than to a one-site model
(figure not shown). From these curves the fractions of
ß1- and ß2-ARs could be calculated as well
as the affinity constant for each receptor subtype. Combining the
results from the saturation and displacement experiments for each
individual allows calculation of the total number of each receptor
subtype. Thus, the increase in total ß-AR density was solely due to
an selective increase in ß2-AR density from about 1
amol/mm2 before to about 2 amol/mm2 after
weight reduction (P < 0.02), whereas the
ß1-AR density was unchanged, being about 2
amol/mm2 before and after weight reduction,
respectively.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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WR resulted in a 50% drop in the basal lipolysis rate, which is in accordance with previous studies on obese subjects not characterized with regard to PCOS (10). Secondly, the lipolytic sensitivity to noradrenaline increased 10-fold in parallel to a 8-fold increase in the ß2-AR subtype sensitivity determined with terbutaline. There was also an increase in isoprenaline sensitivity, which must be due to the ß2-AR effect, as sensitivity to the ß1-AR agonist dobutamine was unchanged. Moreover, this was accompanied by a 2-fold increase in the cell surface density of the ß2-AR, explaining the increase in noradrenaline and terbutaline sensitivities. A low ß2-AR subtype sensitivity has been observed previously in nonobese women with PCOS, upper body obese women, as well as men with the metabolic syndrome (8, 9, 19). In previous studies a slight improvement of ß2-AR sensitivities has been observed after weight reduction of un-selected obese women (10). Weight reduction of obese women with or without PCOS has been shown to improve insulin sensitivity and androgen status as well as lowering the sympathetic tone (20, 21). This was confirmed in the present study. As insulin resistance with high insulin levels are linked to an increased sympathetic activity, both factors could be of pathogenic importance in PCOS (22). In support of an increased sympathetic activity as a primary pathogenic factor in PCOS, both lower ß2-AR subtype density and higher noradrenaline release were found in thecal cells from an experimental rodent PCOS model (23, 24).
Treatment with OC lowered the lipolytic sensitivity to
noradrenaline and isoprenaline due to a selective decrease in the
sensitivity of the ß1-AR subtype. The decrease in
noradrenaline and dobutamine sensitivities could not be attributed to
changes in the ß1-AR, as there was no significant change
in any of the ß-AR subtype densities. Moreover, the effects of
postreceptor-acting agents, such as forskolin and dibutyryl cAMP acting
on the adenylate cyclase and protein kinase C complexes, respectively,
were also unaffected by OC therapy. Likewise, 2-AR
function was unaltered. These findings suggest indirectly a selective
reduction in coupling of the ß1-AR subtype to the
adenylate cyclase as a putative mechanism for the change in
ß1-AR sensitivity, as has been shown in other cell
systems with dual ß-ARs (25). Unfortunately, the small amount of
adipose tissue that was available did not allow ß-AR coupling
experiments. Moreover, estrogens have been shown to lower the lipolytic
rate in white adipocytes from female hamsters through an interaction
with the catalytic unit of the adenylate cyclase, whereas in rodents,
direct effects on Gs protein have been described
(26). The existence of estrogen and progesterone receptors in human
adipocytes has been controversial (27). However, in a recent study both
messenger ribonucleic acid and the estrogen receptor protein were
detected in both genders, with a sex difference in the distribution of
the receptors (28). Women had equal estrogen binding capacity in the
adipose tissue from sc abdominal and visceral depots, whereas men had
twice as high estrogen binding capacity in the sc abdominal as in the
visceral fat depot. These studies support a direct effect of estrogen
on lipolysis tentatively located to the ß1-AR-coupled
Gs protein or its interaction with the catalytic
unit of the adenylate cyclase. Moreover, Cryer and co-workers showed
that estrogen substitution of postmenopausal women lowered the
palmitate flux through a direct action on the adipose tissue (29).
However, further studies are needed to clarify the mechanisms of
estrogen action on adipose tissue lipolysis in humans. OC treatment
lowers the fT level markedly by suppressed ovarian T secretion together
with increased binding protein capacity, which in certain women could
lead to fT concentrations below a critical level needed to maintain a
normal adrenergic function in adipose tissue. In men, it is well known
that a U-shaped relationship exists between insulin resistance and the
fT level (30). An interesting finding is that treatment with the same
dose of OC in lean women with PCOS did not influence the
ß1-AR subtype or any of the other AR subtypes in sc fat
cells (9). This implies that different mechanisms are involved in the
regulation of lipolysis in nonobese and obese PCOS women. As adipose
tissue is the major tissue site for conversion of androgen precursors
to estrogens, a larger adipose tissue mass in addition to lower SHBG
levels in obese subjects result in higher target estrogen
concentrations than those in lean subjects (31).
It is clear that the two forms of intervention to normalize androgen status in obese PCOS have divergent effects. WR improves both insulin sensitivity and catecholamine resistance in the adipocytes’ lipolytic function. Adipocyte catecholamine resistance has been observed in many atherogenic conditions, such as PCOS, obesity, and the insulin resistance (metabolic) syndrome. OC therapy also reduces hyperandrogenicity, but fails to improve insulin sensitivity and intensifies catecholamine resistance in adipose tissue. These findings may have important clinical implications favoring WR over OC as the first line of treatment of hyperandrogenic obese subjects with PCOS.
The WR group was about 10 yr older than the OC group. This may reflect different indications for the choice of treatment among the women. In the younger OC group, infertility was the dominate cause of remittance to the clinic, whereas in the older WR group, other causes, such as hirsutism and bleeding disturbances, were more frequent. However, there is no reason to believe that age has any substantial effect on the observed differences between OC and WR therapies. No other clinical parameters differed between the groups, and lipolysis data were almost identical in the two groups at baseline. Furthermore, age has little influence on lipolytic sensitivity, the chief parameter in present study (32).
In summary, WR improves abdominal adipocyte lipolysis in obese women with PCOS due to an increased ß2-AR density in conjunction with indirect signs of increased insulin sensitivity and decreased sympathetic activity. On the other hand, treatment of PCOS women with OC impairs abdominal adipocyte lipolysis through a desensitization of ß1-ARs not related to density and without signs of influence on sympathetic tone or insulin sensitivity. Both treatments lower the fT level, indicating that hyperandrogenicity is not of importance for lipolysis regulation in obese women with PCOS.
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Acknowledgments |
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Footnotes |
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Received October 9, 1998.
Revised February 24, 1999.
Accepted March 16, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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