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Reproductive Endocrinology |
Department of Medicine, Division of Endocrinology/Metabolism (T.P.C., M.G.H., R.O.-F., J.M.O.), and the Department of Reproductive Medicine (A.J.M., S.C.Y.), University of California-San Diego, La Jolla, California 92093
Address all correspondence and requests for reprints to: Dr. Theodore P. Ciaraldi, Division of Endocrinology (0673), University of California-San Diego, La Jolla, California 92093. E-mail tciaraldi{at}ucsd.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the current report, efforts have been made to further characterize the insulin resistance in adipocytes from obese PCOS and weight-matched normal cycling (NC) women. This study was instigated by the observation that experimentally induced depletion of cellular adenosine in rat adipocytes resulted in an impaired insulin sensitivity of glucose transport stimulation with normal binding (11, 12), a condition that resembles the behavior of adipocytes in PCOS.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eleven women between the ages of 21–36 yr were studied. All
were characterized by ammenorhea or persistent oligomenorrhea of
perimenarchial onset, clinical or biochemical evidence of
hyperandrogenism, and polycystic ovaries documented by ultrasonography.
On the basis of clinical examination, there was no evidence of
acanthosis nigricans. Clinical characteristics are presented in Table 1
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Seven NC women matched for age (range, 28–33 yr) and weight
(Table 1
) served as controls. They were in general good health, with no
clinical evidence of hyperandrogenism. All had 27- to 32-day cycles and
were studied during the early follicular phase (days 2–5) of the
menstrual cycle. Neither PCO nor NC subjects had used any hormonal
preparation during the 3 months preceding the study.
Protocol
The study was approved by the human subjects committee at the University of California-San Diego, and an informed written consent was obtained from each subject. After an overnight fast, subjects were admitted to the General Clinical Research Center at the University of California-San Diego at 0700 h. A rapidly sampled iv glucose tolerance test was performed using previously described methods (13). Insulin was assayed by a double antibody RIA (14). Glucose levels were measured by the glucose oxidase method using a glucose analyzer (Yellow Springs Instrument Co., Yellow Springs, OH). On the following day (between 0800–0900 h) all subjects had fat biopsies taken from the lower abdominal area.
Materials
Biosynthetic human insulin was a gift from Dr. Ron Chance, Eli Lilly Co. (Indianapolis, IN). A14-[125I]Insulin was supplied by Dr. Bruce Frank, also of Eli Lilly Co. 3-O-Methyl-D-[1-14C]glucose ([14C]3-OMG) was purchased from New England Nuclear Corp. (Boston, MA). Collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ), BSA (fraction V) and N6-phenylisopropyl adenosine (PIA) were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN), phloretin was obtained from Biochemical Laboratories (Redondo Beach, CA), and silicone oil was purchased from Union Carbide Corp. (New York, NY). Isoproterenol, pyruvate, and the glycerol determination kit were purchased from Sigma Chemical Co. (St. Louis, MO).
Preparation of human adipocytes
Adipose tissue was obtained by open biopsy of the lower abdominal wall using a previously described method (15). Isolated adipocytes were prepared by a modification (15) of the method of Rodbell (16). After digestion and filtration, the cells were washed four times in a buffer consisting of 150 mmol/L NaCl, 5 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L CaCl2, 2.5 mmol/L NaH2PO4, 10 mmol/L HEPES, and 2 mmol/L pyruvate, pH 7.4, supplemented with 4% BSA. Cells were then resuspended at approximately 5 x 105 cells/mL. Due to the limited amount of tissue available from each biopsy, not all of the studies described below were performed on each subject. The number of subjects assayed is given with each set of results.
Cell counts were performed by a modification of method III of Hirsch and Gallian (17), in which cells were fixed in 2% osmium tetroxide and counted with a model ZB Coulter counter (Coulter Electronics, Hialeah, FL) using a 400-µm aperture tube.
Insulin binding to adipocytes
Isolated cells (
2 x 105 cells/mL) were
combined with [125I]insulin (33 pmol/L) and varying
concentrations of unlabeled insulin (0–18 nmol/L). Binding was
measured after incubation for 1 h at 37 C using a previously
described method (18).
3-OMG transport
Adipocytes (5 x 105 cells/mL) were
incubated at 37 C in the absence or presence of varying concentrations
of insulin (60 min). Transport activity was assessed by measuring
initial rates of uptake of tracer amounts (15–20 µmol/L) of 3-OMG,
using a modification (15) of the method of Whitesell and Gliemann
(19).
Antilipolysis
The antilipolytic effect of insulin was measured as the
inhibition of glycerol release into the buffer. Cells (4 x
105 cells/mL) were first treated for 30 min with or without
isoproterenol (1 µmol/L). Varying concentrations of insulin were then
added. After an additional 60-min incubation, buffer was collected, and
glycerol content was determined by a colorimetric method (20). All
values were corrected for background release measured on cells
processed without any incubation period.
Data analysis
Results are expressed as the mean ± SEM and were compared by Student’s t test for paired or unpaired data, when appropriate, employing the Statview SE+ graphics program. Significance was accepted at P < 0.05 (two-tailed t test). EC50 values for antilipolysis and glucose transport stimulation were obtained by log-logit transformations of individual data.
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Results |
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The clinical and laboratory characteristics of the study subjects
are summarized in Table 1. The groups were matched for age and body
mass index (range, 29–49 for NC and 28–51 for PCOS). Adipose tissue
distribution, as indicated by the waist/hip ratio, did not differ
between groups (0.82 ± 0.02 vs. 0.86 ± 0.02 for
NC vs. PCOS). Adipocyte cell volume, another measure of
obesity, also did not differ between groups (990 ± 127
vs. 1197 ± 99 pL for NC vs. PCOS).
Testosterone and LH levels as well as the LH/FSH ratio were all
elevated in the PCOS group. Fasting glucose levels did not differ
between groups, ruling out overt diabetes, but the PCOS subjects were
hyperinsulinemic, suggesting insulin resistance. This was confirmed by
the response to an iv glucose tolerance test, showing a 50% lower
(P < 0.05) insulin sensitivity index in PCOS (Table 1).
Insulin binding and glucose transport activity
Insulin binding to adipocytes was measured at 37 C under the same conditions as insulin stimulation of glucose transport. Specific binding of a tracer (33 pmol/L) concentration of insulin was not significantly different between the NC (0.73 ± 0.11%/105 cells; n = 7) and PCOS (0.98 ± 0.14; n = 11) groups. Insulin receptor affinity, measured by displacement of labeled insulin, was also the same in cells from both groups (not shown).
Absolute 3-OMG transport activities in the basal state were comparable in cells from both groups (0.35 ± 0.08 vs. 0.27 ± 0.05 pmol/105 cells·10 s in NC and PCOS, respectively). Maximally insulin-stimulated 3-OMG transport rates also did not differ (1.17 ± 0.23 vs. 0.99 ± 0.23). However, PCOS cells had greatly impaired insulin sensitivity (EC50 = 290 ± 42 pmol/L; n = 11) compared to controls (93 ± 14; n = 7; P < 0.005).
Lipolysis
To determine whether responses other than glucose transport were subject to impaired insulin signal transduction, the antilipolytic effect of insulin was studied in adipocytes from NC and PCOS subjects. Basal lipolysis was similar in both groups (0.12 ± 0.03 vs. 0.11 ± 0.02 µmol glycerol/105 cells in NC and PCOS). Isoproterenol stimulated lipolysis to the same level in NC (0.27 ± 0.05) and PCOS (0.24 ± 0.03) cells. Insulin maximally inhibited stimulated lipolysis in NC cells by 40 ± 8%. The maximal antilipolytic effect of insulin in PCOS cells (33 ± 6% inhibition) did not differ significantly from the control value. However, insulin sensitivity for the antilipolytic effect in PCOS subjects (EC50 = 115 ± 33 pmol/L; n = 8) was significantly impaired compared to that in NC cells (42 ± 8; n = 6; P < 0.01).
Adenosine regulation of insulin sensitivity
The characteristics of insulin resistance in adipocytes from PCOS subjects, i.e. normal insulin binding, normal glucose transport activity and responsiveness, and impaired sensitivities for antilipolysis and glucose transport stimulation, are very similar to the conditions created in rat adipocytes by depletion of cellular adenosine (11, 12). Addition of the nonmetabolized adenosine analog PIA to adenosine-depleted rat adipocytes normalizes insulin sensitivity, but has no impact on receptor binding or final transport responsiveness (12). To determine whether cellular insulin insensitivity in PCOS might be related to the observation in rat adipocytes, cells from NC and PCOS subjects were incubated with insulin with or without PIA (2 µmol/L) before transport assay.
Treatment of NC or PCOS adipocytes with PIA had no influence on either
basal or maximally insulin stimulated 3-OMG transport activities (not
shown). Insulin sensitivity of NC cells was not influenced by PIA
treatment (Fig. 1). In contrast, there was a major
effect on the shape of the dose-response curve of PCOS cells, with a
striking PIA-mediated leftward shift of the curve (Fig. 1). This effect
is more quantitatively depicted for each individual EC50
value for five NC and seven PCOS subjects with control and PIA
treatments (Fig. 2). PIA did not influence insulin
sensitivity in NC cells (EC50 = 112 ± 18
vs. 108 ± 20 pmol/L, before vs. after
treatment). In all of the PCOS subjects, a major reduction in
EC50 values occurred, falling from 285 ± 47 pmol/L
before treatment to 70 ± 15 with treatment (P <
0.005).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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There is evidence to suggest that the signaling events that lead to glucose transport stimulation and inhibition of lipolysis diverge at some point in the signaling cascade (22). Adipocytes from the current PCOS subjects displayed normal maximal antilipolytic responsiveness to insulin. This is similar to the situation for glucose transport, where insulin responsiveness was also normal. Interestingly, in PCOS cells, the sensitivity to insulin’s antilipolytic effects was significantly reduced. The 3-fold difference in EC50 values for this response between NC and PCOS cells was the same as that for glucose transport stimulation. This suggests that the site of impaired insulin action in PCOS occurs before divergence of the glucose transport stimulation and antilipolytic pathways, and that the final effector systems responsible for antilipolysis are intact in PCOS.
Depletion of cellular adenosine in rat adipocytes creates a condition with normal insulin binding, normal glucose transport activity, and impaired insulin sensitivity for glucose transport stimulation (11, 12) that resembles the behavior in PCOS adipocytes. Similar observations have recently been made in human adipocytes (23). Insulin sensitivity in adenosine-depleted rat adipocytes can be restored by treatment with a nonmetabolized adenosine analog, PIA (11, 12, 24). We performed a similar manipulation in PCOS adipocytes and showed that PIA addition causes a leftward shift of the dose-response curve for glucose transport stimulation. As such, this effect leads to normalization of insulin sensitivity in these cells. In contrast, adenosine was without effect on the shape of the glucose transport dose-response curve in NC cells.
Studies in rat (24, 25) and human (23, 26) adipocytes have shown that insulin sensitivity for antilipolysis is also impaired by adenosine depletion. This would suggest that the adenosine-dependent event(s) in insulin signaling occur before the pathways for glucose transport and antilipolysis diverge, just as is the case for impaired insulin sensitivity in PCOS adipocytes. From these results it is reasonable to anticipate that PIA treatment of PCOS adipocytes would also normalize insulin sensitivity for antilipolysis. Unfortunately, due to the limited amount of tissue available from each biopsy we were unable to test this hypothesis directly.
Adenosine acts through a cell surface A1 receptor to help maintain a normal state of insulin sensitivity for glucose transport (12, 27). Although the specific steps in the insulin signal transduction cascade that are influenced by adenosine are unknown, the effect appears to involve the efficiency of the coupling mechanisms between the insulin receptor and the glucose transport system. The current results suggest that the mechanism of decreased insulin sensitivity in PCOS cells involves an adenosine-sensitive step, and this could involve alterations in adenosine production and release, expression of specific A1 adenosine receptor subtypes, or the efficiency of A1 receptors coupling through distinct G proteins to pathways that modulate insulin sensitivity. These possibilities are currently under investigation. Evidence in the current report argues against adenosine depletion or the presence of a generalized impairment of adenosine action as the cause of the insulin resistance. Endogenous adenosine maintains a tonic inhibition of adipocyte lipolysis (28, 29, 30). Removal of adenosine results in stimulation of basal lipolysis, often up to levels at which there is no further stimulation by lipolytic agents (23, 28, 30). The normal basal and isoproterenol-stimulated rates of lipolysis in PCOS adipocytes indicate that the perturbation of adenosine signaling in PCOS may be specific for control of insulin sensitivity. There are several physiological conditions under which adenosine sensitivity of adipocytes is regulated. These include lactation (31) and chemically induced hypothyroidism (32), where cells are more sensitive to adenosine. Massively obese humans display a reduced sensitivity to the antilipolytic effects of adenosine (33) that is improved after weight loss (30, 34). As the subjects in the present study were matched for obesity, the later mechanism should not be responsible for the differences between NC and PCOS subjects. Rather, PCOS may represent yet another condition where adenosine signaling pathways are regulated.
The current results give rise to the question of whether the alterations in adenosine modulation of insulin sensitivity are selective for PCOS or occur in other insulin-resistant states. Studies in starved and streptozotocin-diabetic rats, both insulin-resistant conditions, showed no change in the ability of adenosine to regulate the insulin sensitivity of either glucose transport or antilipolysis (35). Any alterations in insulin sensitivity in those conditions could be accounted for by changes in insulin binding. Impaired insulin sensitivity in adipocytes from obese and noninsulin-dependent diabetes mellitus subjects was also found to be due to decreases in insulin binding (36). The obese NC subjects studied in the current report could also be considered insulin resistant compared to lean individuals. Yet, their insulin sensitivity was not influenced by added PIA, suggesting that alterations in adenosine modulation of insulin action are not common to all insulin-resistant states. However, we are unaware of any systematic evaluation of this question, and further studies are necessary to establish whether this particular defect is indeed selective to PCOS.
The identity of the insulin signaling event(s) that is perturbed in PCOS is also an open question. Dunaif et al. have reported on increased serine phosphorylation of insulin receptors isolated from cultured fibroblasts in a subset of PCOS subjects (37). Insulin-stimulated receptor tyrosine kinase activity was also decreased in these subjects. The potential importance of such a difference in receptor kinase activity in fibroblasts is uncertain, because these workers failed to find any correlations between receptor phosphorylation and either in vivo or in vitro insulin action (37). Investigations of cultured fibroblasts obtained from many of the same subjects whose adipocytes were studied in the current report suggested that insulin action was, in fact, normal in fibroblasts (38). In addition, our earlier studies indicated that insulin receptor kinase activity from PCOS adipocytes displayed little or no difference compared to NC adipocytes (8). Considerable additional work will be necessary to identify the crucial steps. Signaling events downstream of the receptor kinase are currently being investigated as potential sites of the impaired insulin sensitivity of PCOS.
Evidence exists suggesting that PCOS can be an inherited disease (39), and therefore, the form of insulin resistance present in these patients may also be genetic. It is possible that the adenosine-sensitive step in PCOS cells represents the site of a genetic abnormality in these patients, and further dissection of the insulin signaling pathways in PCOS may lead to important new insights into the pathogenesis of this common syndrome as well as better understanding of the basic mechanisms of insulin action.
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Acknowledgments |
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Footnotes |
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2 Clayton Foundation Investigator.
Received May 2, 1996.
Revised August 23, 1996.
Revised February 5, 1997.
Accepted February 13, 1997.
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) and PCOS (•) subjects.
EC50 values were determined from dose-response curves for
glucose transport stimulation in control and PIA-treated (2 µmol/L)
cells. Each line connects values from a single
individual.