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Departments of Medicine (G.P., H.O.S., G.H., A.D.B.) and Obstetrics and Gynecology (M.K.S.), Indiana University School of Medicine, Indianapolis, Indiana 46202; and Amylin Pharmaceuticals, Inc. (A.D.B.), San Diego, California 92121
Address all correspondence and requests for reprints to: Alain D. Baron, M.D., Amylin Pharmaceuticals, Inc., 9373 Towne Centre Drive, Suite 250, San Diego, California 92121. E-mail: abaron{at}amylin.com.
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Abstract |
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We studied leg blood flow (LBF) responses to graded intrafemoral artery infusion of the endothelium-dependent vasodilator methacholine chloride (MCh) and to a 4-h hyperinsulinemic euglycemic clamp (120 mU/m2·min) in 10 PCOS, before and after 3 months treatment with Tgz (600 mg/d). A group of 13 obese women (OBW) matched for age, weight, body fat (>40% in both groups), blood pressure, and total cholesterol served as controls.
PCOS patients exhibited elevated free testosterone (fT) and triglycerides (TG) and lower high density lipoprotein cholesterol levels compared with OBW [14.0 ± 1.0 vs. 3.7 ± 0.6 pmol/liter (P < 0.0001), 1.60 ± 0.28 vs. 0.94 ± 0.09 mmol/liter (P < 0.02), and 0.91 ± 0.04 vs. 1.1 ± 0.04 mmol/liter (P < 0.005), respectively]. Tgz treatment reduced fT levels, but did not improve the TG and high density lipoprotein profile [to 9.7 ± 2.8 pmol/liter (P < 0.007), 1.49 ± 0.34 mmol/liter (P = NS), and 0.93 ± 0.07 mmol/liter (P = NS), respectively]. Basal LBF was unchanged after Tgz. In PCOS compared with OBW, insulin stimulated glucose disposal (52.7 ± 6.6 vs. 85.5 ± 4.4 µmol/kg fat-free mass·min; P < 0.0005) and vasodilation (increase in LBF, 22 ± 14% vs. 59 ± 15%; P < 0.05) were significantly improved after Tgz treatment to 68.8 ± 7.2 µmol/kg fat-free mass·min (P < 0.0001) and 101 ± 48% (P < 0.03), respectively. The increase in LBF in response to MCh in PCOS was markedly more pronounced after treatment (P < 0.01, by ANOVA) and was similar to that observed in OBW. Before Tgz treatment, maximal LBF increments in response to MCh were 130 ± 25% and 233 ± 29% in PCOS and OBW, respectively (P < 0.01). After Tgz treatment, PCOS values improved, achieving increments similar to those in OBW (245 ± 45%; P < 0.04).
Tgz treatment in PCOS improves both hormonal and metabolic features. These modifications are associated with improvement of endothelial function, suggesting that Tgz could be a useful tool to reduce the risk of macrovascular disease in women with PCOS and perhaps in other insulin-resistant syndromes.
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Introduction |
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Thiozolidenediones (TZDs) belong to a class of drugs known as peroxisome proliferator activating receptor-
(PPAR) agonists. Although not fully understood, it appears that through their action to alter gene expression these drugs enhance the sensitivity of tissues (fat and muscle) to insulin (15). As such, they are known as insulin sensitizers and are currently used for the treatment of type 2 diabetes characterized by insulin resistance (16, 17, 18). The TZD troglitazone (Tgz) has been reported to ameliorate glucose tolerance, insulin sensitivity, and insulin secretion; partially reverse lipid and fibrinolytic abnormalities in states of insulin resistance; and reduce circulating androgen levels in PCOS (19, 20). In the current study we tested whether treatment with the TZD Tgz of insulin-resistant women with PCOS could ameliorate endothelial function.
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Subjects and Methods |
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Twelve obese (body mass index, >30 kg/m2; body fat, >40%) women with PCOS were recruited for the study between 1997 and 1998. Two of these did not complete the study for personal reasons. Thirteen normal, healthy, glucose-tolerant obese women (OBW) matched with PCOS for age, weight, and body fat served as comparators. Women from both groups were in good health and were not taking medications known to affect carbohydrate or sex hormone metabolism. None of these subjects had hypertension or abnormal oral glucose tolerance tests. Control women had regular menses, no hirsutism, and normal free testosterone (fT) levels. PCOS was diagnosed by an elevation of fT levels, associated with hirsutism and amenorrhea or chronic oligomenorrhea (fewer than six periods per year) (1). Nonovarian causes of androgen excess were not formally excluded. Studies were approved by the Indiana University human subjects internal review board, and all volunteers gave informed consent.
Protocol
All studies were performed in a quiet, temperature-controlled room after an overnight 14-h fast at the General Clinical Research Center of Indiana University Hospital. After the baseline oral glucose tolerance test (OGTT) and dual energy x-ray absorptiometry assessment of body composition, all women underwent both hemodynamic and metabolic studies. Hemodynamic studies of endothelium-dependent vasodilation were performed as described previously (21). In short, a 6 French sheath (Cordis Corp., Miami, FL) was placed in the right femoral vein to allow the insertion of a custom-designed 5 French double lumen thermodilution catheter (Baxter Scientific, Edwards Division, Irvine, CA) to measure leg blood flow (LBF). The right femoral artery was cannulated with a 5.5 French double lumen catheter (Arrow International, Reading, PA) to allow simultaneous infusion of substances through the proximal port (most caudad) and invasive blood pressure monitoring through the distal port (most cephalad). Heart rate and mean arterial blood pressure (MAP) were monitored continuously via precordial leads and a pressure transducer connected to a vital signs monitor (VSM 1, Physiocontrol, Redmond, WA). LBF, MAP, and heart rate measurements were obtained at baseline and during intrafemoral artery infusion of methacholine chloride (MCh) at sequential doses of 5, 10, and 15 µg/min. All hemodynamic measurements were repeated after about 200 min of euglycemic hyperinsulinemia.
Whole body glucose disposal was assessed during a 4-h euglycemic hyperinsulinemic clamp. Each clamp was performed during a square wave infusion of insulin at a rate of 120 mU/m2·min. The serum glucose concentration was maintained at the baseline level by administering a 20% dextrose solution at variable rate according to arterial serum glucose measurements obtained at 5-min intervals. Rates of glucose disposal (GDR) were calculated by averaging the glucose infusion rates achieved over the last 40 min of the clamp.
After baseline studies, women with PCOS underwent therapy with Tgz (600 mg daily) for 3 months, after which they underwent repeat studies of hemodynamic and metabolic variables. Originally, research volunteers returned to the general clinical research center every 4 wk to undergo blood sampling for liver function tests. However, due to the reports of potential liver toxicity of Tgz, we allowed the research subjects to withdraw from the study; none of the volunteers withdrew. However, for safety reasons, liver function tests were checked every 2 wk for the duration of drug treatment. None of the research volunteers experienced elevation of liver function tests above baseline levels.
Analytical methods
Blood for determination of plasma insulin concentrations was collected in tubes treated with heparin. The specimens were spun, and the supernatant was removed and stored at -20 C. Insulin levels were measured using the Coat-a-Count kit, (Diagnostic Products, Los Angeles, CA). Blood for serum glucose determinations was drawn, put into untreated polypropylene tubes, and centrifuged with an Eppendorf microcentrifuge (Brinkmann Instruments, Inc., Westbury, NY). The glucose levels in the supernatants were determined by the glucose oxidase method with a glucose analyzer (YSI 2300 STAT, YSI, Inc., Yellow Springs, OH). Serum total cholesterol and TG levels were measured on a Kodak Ektachem 702 Analyzer by an enzymatic method (Eastman Kodak Co., Rochester, NY). HDL cholesterol was measured with the Magnetic HDL kit (Reference Diagnostics, Inc., Arlington, MA), and low density lipoprotein cholesterol was calculated according to the Friedewald formula (22). Free fatty acid (FFA) was measured according to the method described by Novak (23). Estradiol was determined using double antibody kit (Diagnostic Products). Testosterone (total and free) and dehydroepiandrosterone sulfate were measured with the Coat-a-Count kit (Diagnostic Products). PAI-1 levels were measured by ELISA (Asserachrom PAI-1, American Bioproducts, Parsippany, NJ).
Statistical analysis
Comparison between groups was performed by repeated measurements ANOVA. When significant differences between groups were found by ANOVA, this was followed by post hoc testing with Fisher’s protected least significant difference test. Comparison within a group was performed by paired t test. Because basal LBF differed significantly between groups, changes in blood flow are expressed as the percentage change to adjust for the differences at baseline. Results are shown as the mean ± SEM. Statistical significance was accepted at a level of P value less than 0.05.
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Results |
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Anthropometric characteristics of the study groups are shown in Table 1
. There were no differences between PCOS and OBW in the indexes of body adiposity and fat distribution. Tgz therapy caused a small, but nonsignificant, increase in body weight without changes in percent body fat or waist/hip ratio.
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Glucose and insulin concentrations and whole body glucose disposal rates
Women with PCOS had slightly higher fasting plasma glucose levels than OBW (Table 2). More evident differences were seen in the glucose values during the OGTT, where the increase in glucose concentrations exhibited by PCOS patients was markedly higher than that in OBW (P < 0.001, by repeated measures ANOVA; Fig. 1). After Tgz therapy, glucose values either fasting or postchallenge were dramatically reduced (P < 0.002), showing no difference from those in OBW (P = 0.99; Fig. 1). Fasting plasma insulin concentrations were about 46% higher in PCOS women than OBW (Table 2). Tgz therapy reduced the insulin concentration in women with PCOS by approximately 19% to levels no different from those in OBW (Table 2).
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). Treatment with Tgz improved the whole body insulin sensitivity of women with PCOS by 32% (GDR at steady state, 68.8 ± 7.2 vs. 52.7 ± 6.6 µmol/kg fat-free mass·min; P < 0.0001). However, after therapy, PCOS patients were still more insulin resistant than matched controls (Fig. 2).
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)Women with PCOS had, by design, higher concentrations of free and total testosterone than OBW. PCOS subjects also exhibited lower HDL cholesterol and higher TG and PAI-1 levels than OBW. After Tgz therapy, PCOS subjects showed a remarkable decrease in both free and total testosterone (by 34% and 20%; P < 0.0005 and P = 0.048, respectively). No significant change was observed with regard to HDL cholesterol, whereas there was a slight reduction in TG. PAI-1 values in women with PCOS decreased after Tgz treatment, achieving levels similar to those in OBW.
Hemodynamic data
As shown in Table 2, there were no significant differences in basal MAP values between PCOS and OBW. Tgz caused a small decrease in MAP, but this difference did not reach statistical significance (P = 0.08).
Basal LBF levels were 0.31 ± 0.04 and 0.28 ± 0.02 liter/min in PCOS and OBW, respectively (P = NS). The increments in LBF in response to intraarterial MCh were significantly reduced in the PCOS subjects compared with OBW (P = 0.039, by repeated measures ANOVA; Fig. 3). Maximum LBF increments were 130 ± 25% and 233 ± 29% in PCOS patients and OBW, respectively (P < 0.01). Euglycemic hyperinsulinemia induced a modest LBF rise above baseline in PCOS (22.3 ± 13.9%; P = NS), whereas the increment in LBF in OBW was more marked (58.6 ± 15.5%; P < 0.005). These increments were, in turn, different between the two groups (P < 0.05; Fig. 4). These data indicate that women with PCOS exhibit resistance to the vasodilatory effects of both MCh and insulin.
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). Significant improvement was also seen in the LBF response to hyperinsulinemia, with an increase of 55.4 ± 16.7% (P = 0.032 vs. PCOS before therapy), not different from that observed in OBW (Fig. 4). Thus, treatment with Tgz restored endothelial function and insulin-mediated vasodilation in women with PCOS. |
Discussion |
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agonist Tgz in women with PCOS ameliorated insulin sensitivity, reduced prevailing testosterone and PAI-1 levels, and restored endothelium-dependent vasodilation to control levels. This is the first demonstration of normalization of endothelial function in a clinical state of insulin resistance with Tgz therapy.
Reduced endothelium-dependent vasodilation has previously been reported in various clinical states that carry an increased risk of coronary heart disease, such as hypertension (8) and hypercholesterolemia (10); more recently, insulin resistance (12, 13) has been recognized as such a state. Indeed, reduced vasodilation in response to the muscarinic agonist acetylcholine and insulin (both endothelium-dependent vasodilators) has been demonstrated in common states of insulin resistance such as obesity and type 2 diabetes and in OBW with PCOS (14). Because endothelial dysfunction is thought to represent one of the earliest steps in the development of atherosclerosis (7), it follows that the increased risk of coronary events observed in insulin-resistant states (24, 25, 26) is attributable at least in part to endothelial dysfunction. Indeed, a direct relationship has been observed between the degree of insulin sensitivity and endothelial function, suggesting that insulin sensitivity and endothelial function are closely related (12, 13). If such were the case, therapeutic maneuvers that enhance insulin action would be predicted to ameliorate endothelial function. The recent advent of TZDs for the treatment of insulin resistance in patients with type 2 diabetes allows testing of this idea. These drugs, known as insulin action enhancers or insulin sensitizers, act via binding and activation of the nuclear transcription factor PPAR and the subsequent expression of genes responsible for glucose and fat metabolism (27). TZDs augment insulin-mediated glucose uptake in fat and skeletal muscle without any reduction in body adiposity, a key factor associated with insulin resistance (15, 18). In the current study we confirmed the findings of others that Tgz improves insulin action and reduces PAI-1 and testosterone levels in women with PCOS (19, 20). In addition, we made the novel observation that Tgz therapy ameliorates endothelial function in women with PCOS, as evidenced by achieving methacholine- and insulin-mediated vasodilation equivalent to that observed in obese, non-PCOS controls. Because the latter is entirely nitric oxide dependent (28), it is reasonable to suggest that Tgz therapy enhances nitric oxide release in response to insulin. Although we have not previously found any defects in endothelium-independent vasodilation in other clinical states of insulin resistance, we cannot exclude an effect of Tgz therapy to improve vasorelaxation via an endothelium-independent mechanism.
Metabolic and hormonal changes after Tgz therapy were not accompanied by significant alterations in weight, body fat, cholesterol, or blood pressure, all factors reported to modulate endothelial function (8, 10, 12, 13). Thus, one can conclude that improvement in endothelial function was due to other factors. It is noteworthy that although Tgz therapy enhanced rates of insulin-mediated glucose uptake by 32%, it did not normalize insulin sensitivity. In contrast, Tgz therapy normalized endothelial function compared with that in obese controls, who, compared with lean females, exhibited endothelial dysfunction (29). As our study was not randomized double-blinded, the improvement of endothelial function may have been overestimated. Nevertheless, amelioration or normalization of endothelial function in response to Tgz might indicate that 1) Tgz differentially modulates endothelial function and insulin action; 2) endothelial dysfunction occurs only below a certain threshold of insulin sensitivity; or 3) insulin sensitivity and endothelial function are unrelated. On the other hand, Tgz therapy could have ameliorated endothelial function via reductions in circulating testosterone and/or FFA levels.
The mechanism of action by which Tgz improves endothelial function is not resolved by the current study. The mechanism of action of Tgz on improving insulin-mediated glucose uptake in skeletal muscle is also not clear, as PPAR receptors are predominantly found in adipocytes. One prevailing hypothesis is that TZDs reduce lipolytic rates and thus FFA flux from adipocytes to muscle, which improves insulin action in that tissue. Indeed, because elevation of FFA can cause both insulin resistance (30) and endothelial dysfunction (31), this is an attractive proposition. Although we found an effect of Tgz to reduce basal circulating FFA levels, this change was not statistically significant, but this finding does not rule out a decrease in postprandial or daylong prevailing FFA levels. Recent reports indicate that insulin activates the endothelium-based nitric oxide synthase via a signal transduction system (32, 33) very similar to that insulin uses to activate glucose transport in adipocytes and skeletal muscle cells. Thus, if Tgz improves any aspect of insulin signaling, one might expect coordinate improvements in both endothelial function and insulin-mediated glucose transport in insulin-resistant states. Finally, Tgz may modulate reactive oxygen species generation (34), endothelin secretion (35), or vascular inflammation (36), which would improve vascular function. As each of the different TZD’s activates different sets of genes, the results of our study may not apply to the other TZDs, such as rosiglitazone or pioglitazone. On the other hand, if the mechanism of endothelial function improvement is related to amelioration of insulin sensitivity, one might expect that these results may be generalizable to the TZD class.
It is important to consider the clinical implications of our findings for patients with PCOS and potentially for all states of insulin resistance. Insulin resistance is common and is estimated to affect up to 30% of our population. Moreover, it is now recognized that insulin resistance is associated with an increased risk of cardiovascular disease (25, 26). Given that endothelial dysfunction is considered to be an early and necessary abnormality in the pathogenesis of atherosclerosis and coronary disease, it follows that amelioration of endothelial function in states of insulin resistance holds the promise of reducing cardiovascular disease events. Although our data support this idea in patients with PCOS, it remains to be determined for other insulin-resistant states. True outcome trials will be required to definitively address this issue, but it is intriguing to speculate whether TZD therapy may have a role in the prevention of cardiovascular disease independent of its effects on prevailing plasma glucose concentrations in patients with insulin resistance and type 2 diabetes.
In summary, we have shown that treatment with the TZD Tgz for 3 months in patients with PCOS improves endothelial function. This effect is probably related to the drug’s effect to reverse insulin resistance, and thus TZD therapy may have a role in the prevention of cardiovascular disease.
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Acknowledgments |
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Footnotes |
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G.P. is the recipient of a grant from the A. Griffini Foundation (Varese, Italy).
Abbreviations: FFA, Free fatty acid; fT, free testosterone; GDR, glucose disposal rate; HDL, high density lipoprotein; LBF, leg blood flow; MAP, mean arterial blood pressure; MCh, methacholine chloride; OBW, obese women; OGTT, oral glucose tolerance test; PAI-1, plasminogen activator inhibitor-1; PCOS, polycystic ovary syndrome; PPAR, peroxisome proliferator activating receptor-; TG, triglycerides; Tgz, troglitazone; TZD, thiazolidenedione.
Received March 12, 2002.
Accepted October 17, 2002.
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) and after treatment (
) during a 3-h OGTT.
%) in response to graded intrafemoral artery infusions of MCh in obese control women (•) and obese PCOS subjects before (