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Original Article |
Department of Obstetrics and Gynecology (F.C., D.R., G.M., S.M.), Catholic University of Sacred Heart, 00168 Rome, Italy; and OASI Institute for Research (L.S., A.L.), 94018 Troina (Enna), Italy
Address all correspondence and requests for reprints to: Antonio Lanzone, M.D., Department of Obstetrics and Gynecology, Catholic University of Sacred Heart, L. go A. Gemelli 8, 00168 Rome, Italy. E-mail: . alanzone{at}rm.unicatt.it
Abstract
The effect of raloxifene, a selective estrogen receptor modulator recently approved as a therapeutic agent for menopause, on glyco-insulinemic metabolism was investigated in 40 healthy postmenopausal women.
At the baseline and after 12 wk of raloxifene (60 mg/d) or placebo administration, all aspects of glucose metabolism were evaluated in each subject using both an oral glucose tolerance test (OGTT; 75 g) and a hyperinsulinemic euglycemic clamp to assess peripheral insulin sensitivity. Glucose, insulin, and C-peptide, measured in fasting conditions, as well as glucose and insulin responses to OGTT [expressed as area under curve (AUC)] were not modified by raloxifene, whereas C-peptide-AUC increased significantly (P < 0.05). Furthermore, a trend toward an improvement of peripheral insulin sensitivity and hepatic clearance of the hormone (fractional hepatic insulin extraction) was observed in the raloxifene-treated women with respect to the control patients. When the subjects were studied in relation to their insulin secretion in response to the glucose load, the patients, classified as hyperinsulinemic, showed the most significant response to the raloxifene treatment. In these women, the selective estrogen receptor modulator was able to induce a significant reduction of insulin circulating plasma values (P < 0.01) through both an increase of fractional hepatic insulin extraction (P < 0.01) and an improvement of the peripheral insulin sensitivity (P < 0.05). On the contrary, no net change of insulin dynamics was observed in normoinsulinemic and placebo-treated women.
The present data indicate that raloxifene does not negatively influence glyco-insulinemic metabolism in unselected postmenopausal women and may indeed improve the excessive insulin responsiveness to OGTT in a selected population of hyperinsulinemic postmenopausal women.
THERE ARE FEW studies of the effects of menopause, either surgical or natural, on glucose metabolism and insulin action; earlier data suggested a deterioration in glucose tolerance and an increase in plasma insulin (1), whereas other investigations found no significant changes in the carbohydrate metabolism (2). The most detailed evaluation to date (3) indicated that the menopause status, per se, is related to a reduced pancreatic insulin response to glucose and a diminished rate of insulin elimination. Hence, the deficiency in the insulin secretion is compensated by a reduced elimination, which results in no net effect on either glucose tolerance or insulin plasma concentrations. When insulin sensitivity was investigated, as a result of the standardization of the studied population or the different mathematical modeling analysis used, conflicting data were found. Detailed evaluation by Lindheim et al. (4) and Godsland et al. (5) revealed a negative correlation between the age of menopause and the insulin sensitivity or the hepatic insulin elimination, or both.
Although recent data from the Heart and Estrogen/ Progestin Replacement Study showed no apparent effect of hormone replacement therapy (HRT) on cardiovascular end-points in postmenopausal women with established coronary heart disease (6), many observational studies provide evidence of lower rates of primary and secondary cardiovascular adverse events in women taking postmenopausal estrogens than in women not receiving this therapy (7, 8). Concerning this, it is believed that the estrogen benefits may be ascribed to several mechanisms, such as the restoration of the endothelium-dependent vasodilation of coronary arteries and the favorable changes in plasma lipids (9, 10).
In vivo and in vitro studies (11) put in evidence that the link between the hypothesized beneficial effect of estrogen replacement therapy (ERT) and the suggested effect on the lipid pattern may be insulin secretion and/or insulin sensitivity or glucose-induced reuptake, which are clearly involved in the pathophysiological mechanism of noninsulin-dependent diabetes mellitus, hypertension, and coronary heart disease. In our previous studies, we demonstrated that ERT and estro-progestin combined therapy (HRT) were able to improve insulin sensitivity and reduce insulin plasma levels, above all, in hyperinsulinemic postmenopausal population, thus leading to a significant improvement in the glyco-insulinemic homeostasis and a supposed improvement of the cardiovascular disease risk (12).
Although a large body of data supports the above-mentioned effect of ERT, the increased risk of cancer in reproductive tissue (breast and endometrium) adversely affected the compliance for long-term treatment (13, 14, 15). To date, raloxifene, a selective estrogen receptor modulator (SERM), seems to be a potentially viable alternative to ERT and HRT. Raloxifene exerts tissue-selective effects similar to that of estrogen on bone (16, 17) and serum lipids in both rodents (18) and humans (19) without any apparent stimulatory effect in cell culture or in vivo on mammary (20, 21) and uterine tissues (18, 22, 23, 24). There are few published data on the effect of raloxifene or other SERMs or antiestrogens, such as tamoxifen, on glyco-insulinemic metabolism in postmenopausal women. Here, we report the effects of raloxifene on glyco-insulinemic metabolism in a placebo-controlled study with healthy postmenopausal women.
Subjects and Methods
Subjects and study design
After screening, 40 Caucasian postmenopausal women aged 47–63 yr (52.8 ± 0.95; mean ± SEM) who were attending the gynecological department of our university for the relief of menopausal symptoms were submitted to the study protocol. Women were 6.5 ± 1.2 (mean ± SEM) yr postmenopausal; none had undergone a hysterectomy or a bilateral oophorectomy. Before beginning the study, assessment of the plasma FSH (>35 IU/liter) and 17-ß estradiol (E2; <73 pmol/liter) concentrations, a mammography, a cervical cytology, and a transvaginal ultrasound examination of the ovaries and the endometrial thickness was performed. The above-mentioned parameters were found to be normal or compatible with menopausal status. No patient was currently taking drugs known to affect lipid or glucose metabolism; none of them had taken any steroids within the previous 6 months. None smoked more than 10 cigarettes per day or drank more than 300 g of alcohol per week. Diabetes or impaired glucose tolerance, breast cancer, liver or kidney parameter alterations, history of major thromboembolism, thyroid disease, and uncontrolled or treated hypertension (systolic blood pressure
160 mm Hg or diastolic 90 mm Hg) were considered as exclusion criteria. The study was approved by the Ethical Review of our University, and an informed consent was obtained from each woman.
On the basis of a balanced computer-generated randomized design, stratified for insulin-AUC (area under the curve) after oral glucose tolerance test (OGTT), eligible subjects who agreed to participate in our prospective placebo-controlled study were randomly allocated to one of the two treatment groups: a group treated with raloxifene hydrochloride, 60 mg once daily (Eli Lilly \|[amp ]\| Co., Indianapolis, IN) and a placebo group. Active drugs and placebo were superimposable in appearance, and both were administered for 12 wk.
Measurements
Women were hospitalized before and after the treatment. During each hospitalization, FSH, LH, E2, estrone, deydropiandrosterone- sulfate (DHEA-S), androstenedione, 17-hydroxyprogesterone, testosterone, and SHBG were assayed. Furthermore, the metabolism of carbohydrates was studied by an OGTT and a hyperinsulinemic euglycemic clamp. The tests were performed on two different days in a randomized order.
After following a standard carbohydrate diet (300 g/d) for 3 d and fasting overnight for 10–12 h, the patients underwent the metabolic evaluation.
During OGTT, basal blood samples were collected at time 0 and 30, 60, 90, 120, 180, and 240 min after the ingestion of 75 g glucose in 150 ml water. Glucose, insulin, and C-peptide were assayed for each sample.
On a different day, a hyperinsulinemic euglycemic clamp was performed to assess peripheral insulin sensitivity. A retrograde intravenous catheter was inserted into a forearm vein for blood sampling and kept in a warming device at 60 C to arterialize the venous blood samples. Another indwelling catheter was inserted into the controlateral forearm vein (Cavafix, B. Braun, Melsungen, Germany) for the glucose and insulin ingestion. A two-step primed contact infusion of human insulin (Actrapid HM, Novo Nordisk Pharma Ltd., Copenhagen, Denmark) was administered at a rate of 40 mIU x m2 x min-1. After reaching the steady state of velocity of insulin infusion within 10 min to achieve the steady-state insulin circulatory concentrations of about 717 pmol/liter during the clamp (range, 574–897 pmol/liter), a variable infusion of 20% glucose was initiated via separate infusion pumps. Blood samples were taken every 5 min from the arterialized line, and blood glucose infusion was adjusted according to a standard algorithm to maintain the blood glucose level between 4.4 and 4.9 mmol/liter.
Samples for plasma glucose concentrations, as well as for biochemical parameters, were assayed immediately. For all other determinations, samples were promptly centrifuged, and the plasma was stored at -20 C until assayed. All hormones were measured by a commercial RIA (Radim, Pomezia, Italy). The intra-assay and interassay coefficients of variation were less than 8% and 15%, respectively, for all hormones. Plasma glucose concentrations were determined by the glucose-oxidase method with a glucose analyzer (Beckman Instruments Inc., Palo Alto, CA). A normal glycemic response to OGTT was defined according to the criteria of the American Diabetes Association (25).
Insulin and C-peptide plasma levels were expressed as fasting value and as AUC after the glucose load, which was calculated by the trapezoidal rule. The patients were classified as normoinsulinemic and hyperinsulinemic according to their insulin response to OGTT, assuming an AUC cut-off value of 107,625 pmol/liter x 240 min (12, 26), which was calculated by using the mean + 2 SD for about 100 OGTTs that were performed in control lean subjects and confirmed by a cluster analysis.
Pancreatic insulin secretion was analyzed on the basis of C-peptide concentrations, because of its equimolar secretion with insulin and its poor and negligible hepatic metabolism (27). Fractional hepatic insulin extraction (FHIE) was estimated by the difference between the incremental AUC of C-peptide and insulin and the incremental C-peptide AUC (28). The incremental area for both C-peptide and insulin was obtained from the difference between AUC and basal AUC (basal AUC = area of the curve due to fasting value x 240 min). Insulin sensitivity was calculated as total body glucose utilization [metabolic index, (M)], set between 60 and 240 min of the glucose clamp and expressed as milligrams per kilogram of body weight x minutes-1, because the M/insulin ratio fails to narrow the range of the individual sensitivity values (29).
Transvaginal ultrasound measurement of the long axis, double layer endometrial thickness, and uterine dimension were performed with an endovaginal probe (>5 MHz). The maximum endometrial thickness (millimeters) was measured after identification of the uterus in a mid-longitudinal plane on the basis of the cervical canal.
Body mass index (BMI) was evaluated according to the ratio of weight (kilograms) to height (meters2). Patients with a BMI of 25 or greater were defined as obese.
Waist (W) to hip (H) ratio (WHR) was used to define body fat distribution (W is circumference obtained from the minimum value between the iliac crest and the lateral costal margin; H is circumference of the minimum value over the buttocks) (30). Women with WHR values greater than 0.85 were considered to have an abdominal (android) fat distribution.
Statistical analysis
Data were stored and analyzed using SPSS software (release 6.0; SPSS, Inc., Chicago, IL) on an IBM-compatible computer. The Kolmogorow-Smirnov test was performed to assess the differences in the general shapes of the distributions. Not normally distributed variables were logarithmically transformed. For within-group changes, tests with the use of two-tailed t test for paired data were done, whereas comparisons between the groups were made by a one-way ANOVA; any significant differences were identified using the Bonferroni correction for multiple comparison. The relationship among the variables was analyzed using the linear regression analysis. P values less than 0.05 were considered statistically significant. All results are expressed as mean ± SE.
Results
All patients who were recruited completed the study protocol. No adverse events like recurrence of hot flashes or leg cramps were registered in either the raloxifene group (21 patients) or the placebo control group (19 patients). No developed skin irritation, bleeding disturbance, or mastalgia was observed. Blood pressure was stable during the 3 months of the study, and no difference in glomerular filtration was found between the two groups and in relation to treatments. General features, like age (55 ± 1.3 vs. 54 ± 1.6 yr), menopausal age at recruitment (7 ± 0.9 vs. 6.3 ± 1.8 yr), BMI (27.8 ± 1.3 vs. 26.9 ± 1.2 kg/m2), WHR (0.85 ± 0.01 vs. 0.84 ± 0.01), and endometrial thickness (3.5 ± 2.5 vs. 3.3 ± 2 mm) were comparable between raloxifene-treated and control patients and not significantly different before and after treatment.
Table 1
indicates the hormonal and metabolic characteristics of the two groups in the baseline conditions and after 12 wk of raloxifene or placebo administration.
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According to their insulin response to OGTT, 9 women (43%) were then classified as hyperinsulinemic (H) and 12 (57%) as normoinsulinemic (N) in the raloxifene group; 8 women (42%) were classified H and 11 (58%) were classified N in the control group.
In Table 2 are reported the clinical and hormonal features of these two subgroups of patients, before and after raloxifene or placebo treatment.
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At baseline in the raloxifene-treated group, fasting glucose and C-peptide plasma concentrations were not different between H and N patients, whereas fasting insulin plasma levels were significantly higher in the H group (101 ± 23.3 vs. 52.3 ± 15.9 pmol/liter; P < 0.05). After raloxifene treatment, glucose and insulin plasma levels remained unchanged, whereas C-peptide increased in all patients, but the difference in comparison to baseline value reached the statistical significance only in the H patients (496.5 ± 191.1 vs. 827.5 ± 190.5 pmol/liter; P < 0.01). No change in these parameters was observed in the control group.
Figure 1 shows the effect of raloxifene therapy on ß-cell activity, insulin hepatic extraction, and insulin sensitivity only in H patients.
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Figure 2 shows the effect of raloxifene on the N group in the above parameters.
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Finally, a significant inverse linear correlation was found between M values and C-peptide ß-cell production only before treatment (r = -0.85; P < 0.001), whereas the increase in percentage of SHBG (
SHBG) in raloxifene-treated women showed a direct positive linear correlation with the increase in percentage of FHIE (r = 0.58; P < 0.007).
Discussion
Many published studies have evaluated the influence of estrogen treatment on glucose metabolism; many of these studies were consistent with improved insulin sensitivity in the women taking estrogen alone (31). Our previous data have shown that physiological transdermal substitution alone or with progestin addition may potentially improve insulin secretion, metabolism, and also the peripheral insulin sensitivity in a selected hyperinsulinemic postmenopausal population (12). A minority of postmenopausal women currently take estrogen or HRT long enough for maximum benefits, because of the side effects such as vaginal bleeding and breast pain and because of the fear of uterine and breast cancer (13, 14, 15). Raloxifene, a nonsteroidal benzothiofene classified as a SERM, represents a structurally diverse compound that interacts with the estrogen receptors but elicits agonist or antagonist activity depending upon the tissue type. Raloxifene produces estrogen-like effects on bone and cholesterol metabolism and is estrogen antagonistic in the uterus and breast. SERMs like raloxifene, which have an improved safety profile in reproductive tissues, represent a potentially important alternative to HRT in postmenopausal women for the prevention and treatment of osteoporosis and cardiovascular disease. In preliminary clinical studies, a dosage ranging from 50–600 mg seems to decrease bone turnover and lower serum cholesterol concentration without increasing triglycerides and without any stimulation on the endometrium (32, 33). Moreover, antitumor properties of raloxifene were demonstrated in treated postmenopausal women compared with placebo; after 4 yr of therapy, a reduced risk of breast cancer was reported, whether as a consequence of the prevention of new cancers or the suppression of subclinical tumors, or both (34).
Based on our above observations on the differential effects of estrogens on glyco-insulinemic metabolism, we wanted to investigate the effect of raloxifene on such parameters.
Although there was an increase of pancreatic C-peptide production, the net result of the current study was that raloxifene therapy apparently did not affect either fasting and stimulated insulin and glucose plasma levels or insulin sensitivity in unselected healthy postmenopausal women. This finding seems to confirm recent published data in postmenopausal women with type 2 diabetes mellitus who fail to show an effect of raloxifene on fasting blood glucose, fasting insulin, and insulin sensitivity (35), whereas another study, in older postmenopausal women with osteoporosis, indicates moderately beneficial effects on these parameters (36). In any case, aside from the apparent neutral effect of raloxifene illustrated here, to date there are no data showing whether raloxifene or other SERMs affect glycemic control or insulin sensitivity in postmenopausal women without metabolic disease. The most relevant clinical finding of the present study is that raloxifene had differential effects on insulin metabolism in relation to the metabolic status of these women. In patients showing an exaggerated insulin response to the OGTT, classified as hyperinsulinemic, 12 wk of raloxifene treatment were able to reduce such insulin response up to about 35%; this effect was due to a 3- to 4-fold increase of FHIE despite a concomitant increase in ß-cell activity, as indicated by the augment of C-peptide-AUC. Moreover, an increase of peripheral insulin sensitivity was observed, which in turn was able to counteract the decrease of the insulin circulating levels in the light to achieve euglycemia. On the contrary, in the normoinsulinemic group, raloxifene did not affect any of the above-mentioned parameters. Even if a slight increase of C-peptide ß-cell production was observed, it was counteracted by an equally slight and not significant increase of FHIE, which in turn maintained the insulin plasma concentration unchanged. To date, to our knowledge, no data were published about this issue.
Some studies indicate that HRT may have moderately beneficial effects on insulin sensitivity or secretion and glucose homeostasis (31, 37, 38). As we have previously shown, ERT and HRT were able to exert a positive effect on glucose homeostasis in postmenopausal women by fully displaying their effect on insulin secretion, metabolism, and peripheral sensitivity above all in the hyperinsulinemic patients. Therefore, the bulk of our findings indicate that raloxifene produces preeminent estrogen agonism with respect to glyco-insulinemic metabolism. Taken together, these results demonstrate tissue selectivity for expression and/or degree of estrogen agonism vs. antagonism by raloxifene at hepatic, pancreatic, and peripheral sites. In this concern, poor data are available on tamoxifen (39, 40); although a wide variety of intermediate markers of cardiovascular risk are favorably influenced by tamoxifen, like a reduction of total or low-density lipoprotein cholesterol, a significant beneficial effect of tamoxifen on cardiovascular outcomes is not supported by currently available clinical evidence (41). On the other hand, no data are available on the effect of tamoxifen on glucose homeostasis.
The hypothesis of an estrogen-like mediated action of raloxifene on several metabolic parameters is supported by the observation that, in animals and in humans, raloxifene has a hypocholesterolemic activity via interaction with the estrogen receptor as shown in vitro and in vivo (32, 33, 39). These results are confirmed by our study in which a significant decrease of low-density lipoprotein cholesterol was achieved after 12 wk of treatment (data not shown). This hepatic estrogen-like effect is confirmed by the induced increase in SHBG levels, clearly displayed in the hyperinsulinemic group as well as the normoinsulinemic group, irrespective of BMI.
Moreover, these results together with the concomitant neutral action of raloxifene on testosterone, DHEA-S, and other androgens indicate an improvement of the baseline hyperandrogenicity in postmenopausal women. It is possible that, in the present clinical trial, the above-mentioned action of the SERM on glyco-insulinemic homeostasis might be partially mediated by the effect on circulating androgen levels. To consolidate this hypothesis, hyperinsulinemic postmenopausal women, characterized by a prevalent central (android) body fat distribution, showed the more marked response to raloxifene treatment. Data by Andersson et al. (35) reported a similar effect of raloxifene, without any change in glyco-insulinemic homeostasis. However, this study was conducted in postmenopausal women with type 2 diabetes mellitus, and many of these women were in treatment with oral hypoglycemic agents that might have invalidated the results of raloxifene effect on peripheral insulin action. In any case, the relationship among hyperinsulinemia and hyperandrogenicity is not completely understood. Hyperinsulinemia increases androgen output from the ovary (42) and may suppress SHBG production in the liver (43, 44), suggesting that hyperinsulinemia causes hyperandrogenicity (45). Moreover, previous studies showed that reducing androgens does not improve insulin resistance (37).
In conclusion, this is the first report in which it is shown that raloxifene may potentially reduce insulin responses to OGTT in healthy postmenopausal women, above all in those women indicated as hyperinsulinemic. Probably this mechanism should be taken into account in a larger study sustaining the idea that raloxifene treatment may reduce, by acting through glyco-insulinemic balance, these mentioned cardiovascular risk factors in humans.
Acknowledgments
Footnotes
Abbreviations: BMI, Body mass index; DHEA-S, deydropiandrosterone-sulfate; E2, 17-ß estradiol; ERT, estrogen replacement therapy; FHIE, fractional hepatic insulin extraction; H, hyperinsulinemic; HRT, hormone replacement therapy; M, metabolic index; N, normoinsulinemic; OGTT, oral glucose tolerance test; SERM, selective estrogen receptor modulator; WHR, waist to hip ratio.
Received August 8, 2001.
Accepted June 13, 2002.
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, P < 0.05; 