- Email: [email protected]
Effects of raloxifene on insulin sensitivity, β-cell function, and hepatic insulin extraction in normal postmenopausal women
Effects of raloxifene on insulin sensitivity, b-cell function, and hepatic insulin extraction in normal postmenopausal women Manubai Nagamani, M.D.,a Alexandria Szymajda, M.D.,b Vicken Sepilian, M.D.,a Randall J. Urban, M.D.,b and Charles Gilkison, RN, F.N.P.b a Department of Obstetrics and Gynecology, and Galveston, Texas
b
Department of Internal Medicine, University of Texas Medical Branch,
Objective: To investigate the effect of raloxifene on insulin sensitivity, b-cell function, hepatic insulin clearance, and glucose tolerance in postmenopausal women. Design: Prospective study. Setting: University of Texas Medical Branch at Galveston, Texas. Patient(s): Twenty normal postmenopausal women. Intervention(s): An oral glucose tolerance test (OGTT) was performed on all study participants before and after treatment with 60 mg of raloxifene daily for 3 months. Blood samples were obtained at baseline and 1, 2, and 3 hours after 75-g oral glucose administration for measurement of glucose, insulin, proinsulin, and c-peptide levels. Insulin tolerance test (ITT) and euglycemic clamp studies were also performed before and after treatment. Main Outcome Measure(s): Glucose and insulin area under curve (AUC) were calculated. The c-peptide to insulin ratio was determined to assess hepatic clearance of insulin. The homeostasis model assessment (HOMA) was used to calculate the index of insulin resistance (HOMA-IR) and b-cell function (HOMA-%b). Insulin sensitivity was assessed by insulin tolerance test and glucose infusion rate (GIR) during euglycemic clamp studies. Result(s): There was no change in fasting or AUC glucose levels. Fasting insulin levels were not statistically significantly different, but the insulin levels at 2 hours and insulin AUC were higher after treatment compared with before treatment. Proinsulin, c-peptide levels, and HOMA-%b did not change. The c-peptide to insulin molar ratio was statistically significantly decreased after treatment. There was no change in insulin sensitivity. Conclusion(s): These results indicate that raloxifene has no adverse effect on insulin sensitivity or glucose tolerance, and it does not affect b-cell function. After glucose load, raloxifene decreases hepatic insulin extraction and thus conserves insulin, which may be beneficial to patients with decreased b-cell reserve or those predisposed to type 2 diabetes. (Fertil Steril 2008;89:614–9. 2008 by American Society for Reproductive Medicine.) Key Words: Raloxifene, insulin sensitivity, b-cell function
There is a progressive increase in insulin resistance with aging and, hence, an increase in circulating insulin levels in postmenopausal women (1, 2). Poehlman et al. (3) found that postmenopausal women have increased fasting insulin levels compared with premenopausal women. Insulin resistance and associated metabolic syndrome lead to an increased risk of coronary heart disease and type 2 diabetes (4, 5). The presence of diabetes further increases the risk for fatal ischemic heart disease in women (6, 7). Previous studies of the effect of hormone therapy (HT) on insulin resistance in postmenopausal women have been controversial (8). Studies in primates and humans indicate that progestational agents reduce insulin sensitivity and dilute the beneficial effects of estrogen (9, 10). Raloxifene is a selective
estrogen receptor modulator that is approved for prevention and treatment of osteoporosis (11). It acts as estrogen receptor agonist in bone tissue, and as an estrogen antagonist in breast tissue and endometrium (12). One of the benefits of taking raloxifene instead of HT is that there is no need for additional progestational agents. Raloxifene does not affect insulin sensitivity or glycemic control in postmenopausal women with type 2 diabetes (13).
Received January 17, 2007; revised and accepted March 27, 2007. Supported in part by a grant from Eli Lilly and the National Institutes of Health GCRC M01 RR00073. Presented at the Endocrine Society Annual Meeting; San Diego, California; June 2005. Reprint requests: Manubai Nagamani, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology; University of Texas Medical Branch, Galveston, TX 77555-0587 (FAX: 281-486-5554; E-mail: [email protected]).
MATERIALS AND METHODS Patients Twenty normal postmenopausal women (45 to 62 years of age) who had their last menstrual period at least 1 year before study enrollment and had not taken any HT for at least 3 months before enrollment were included in the study. Inclusion criteria consisted of a follicle-stimulating hormone
614
The purpose of our study was to investigate the effect of treatment with raloxifene for 12 weeks on insulin sensitivity, glucose tolerance, b-cell function, and hepatic insulin clearance in normal postmenopausal women.
Fertility and Sterility Vol. 89, No. 3, March 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.03.083
(FSH) level of >40 mIU/mL and serum estradiol levels of <40 pg/mL. Women with a history of thromboembolic disease were excluded from the study. Women with any major medical illnesses, including clinically significant abnormal liver and kidney function tests, untreated thyroid disease, or diabetes mellitus, were also excluded. The study was approved by the institutional review board of the University of Texas Medical Branch (UTMB), Galveston, Texas. All participants signed an informed consent before inclusion into the study. Clinical data on the patients studied are shown in Table 1. All women maintained a 300-g carbohydrate diet for 3 days before taking an oral glucose tolerance test (OGTT). After overnight fasting, all patients were admitted to the Clinical Research Center (CRC) at UTMB. A standard OGTT, insulin tolerance test (ITT), and euglycemic clamp studies were performed on all participants. Fasting blood samples were also obtained for total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride levels. All of the women were then started on raloxifene, 60 mg daily for 12 weeks. During the last week of treatment, the patients were readmitted to the CRC, and all of the tests were repeated. Oral Glucose Tolerance Test The insulin, c-peptide, and proinsulin levels at fasting and in response to oral glucose challenge were assessed. All of the women maintained a 300-g carbohydrate diet for 3 days before the OGTT. Blood samples were obtained at fasting and at 1, 2, and 3 hours after glucose ingestion (75 g) for measurement of glucose, insulin, proinsulin, and c-peptide levels. Insulin resistance and b-cell function were assessed by homeostasis model assessment (HOMA) from fasting glucose and insulin or c-peptide concentrations (14). The HOMA values for insulin resistance (HOMA-IR) and percentage b-cell function (HOMA-%b-cell) were calculated as previously described elsewhere (14), using a HOMA computer program (HOMA 2 calculator V2.2; The University of Oxford, Diabetes Trials Unit, Headington, Oxford, UK). An ideal normal-weight individual, aged <35 years, has a HOMA-IR of 1.0 mol mU/L2 and a HOMA-%b-cell function of 100%. The c-peptide to insulin molar ratio was determined to assess hepatic insulin extraction. Euglycemic Clamp Studies Single-dose euglycemic clamp studies were performed using modifications to the method described by Rizza et al. (15). The details of our technique have been previously described elsewhere (16). Briefly, after an overnight fast, the women were maintained at bedrest until completion (approximately 6 hours). Three intravenous lines were inserted, including one for infusions, one for sampling, and one for constant withdrawal for continuous blood glucose readout using a Biostator monitor (Life Science Instruments, Miles Laboratories, Elkhart, IN). After a 90-minute baseline infusion period, four plasma samples were drawn at 10-minute intervals. At 2 hours, a primed insulin infusion of 40 mU/m2 per minute was Fertility and Sterility
TABLE 1 Metabolic and endocrine characteristics of postmenopausal women before and after raloxifene treatment. Raloxifene treatment Before
After
BMI (kg/m2) 28.6 1.5 28.6 1.4 FSH (mIU/mL) 71.8 4.6 62.2 4.6 Estradiol 14.4 2.5 13.8 2.2 (pg/mL) Triglycerides 113.5 11.4 116.6 14.3 (mg/dL) Cholesterol 198.4 7.5 185.1 5.7 (mg/dL) HDL (mg/dL) 54.4 58.1 55.9 4 LDL (mg/dL) 120.9 6.4 106.6 3.8 HbA1C 5.5 0.1 5.5 0.1
P value NS < .04 NS NS .027 NS .006 NS
Note: BMI, body mass index; FSH, follicle-stimulating hormone; HbA1C, hemoglobin A1C; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NS, not statistically significant. Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
begun and continued for 3 hours. Euglycemia (85 mg/dL) was maintained by variable infusion of 20% glucose, which was modulated based on blood glucose concentrations and continuously monitored with the Biostator monitor. During the last 30 minutes of the insulin infusion period, four plasma samples were obtained at 10-minute intervals for glucose and insulin measurements. The whole body glucose utilization at the achieved insulin level was quantitated as the mean glucose infusion rate (GIR) during the last 30 minutes of the infusion. Insulin Tolerance Test Responsiveness to exogenous insulin was evaluated by determining the rapidity of fall in plasma glucose (KITT) after an intravenous bolus of insulin of 0.1 units/kg body weight. Human insulin was diluted to a concentration of 1 unit/mL with the diluent provided by Eli Lilly (Indianapolis, IN). The plasma glucose values at 5, 10, 15, and 20 minutes were used to calculate (KITT). Hormone Assays Blood glucose was assayed by the glucose oxidase method (Beckman Glucose Analyzer, Beckman Instruments, Fullerton, CA). Insulin levels were measured by radioimmunoassay kits obtained from Diagnostic Systems Laboratories (Webster, TX). Proinsulin and c-peptide levels were measured by radioimmunoassay with kits purchased from Linco Research (St. Charles, MO). Serum triglycerides, cholesterol, and HDL levels were measured using specific enzyme methods. 615
TABLE 2 Insulin and glucose levels during oral glucose tolerance test and insulin resistance studies in postmenopausal women before and after raloxifene treatment. Raloxifene treatment OGTT Fasting insulin (mU/mL) 2-hour insulin Insulin AUC Fasting glucose (mg/dL) 2-hour glucose Glucose AUC ITT Euglycemic clamp GIR (mg/kg/min) HOMA-IR HOMA-IR insulin HOMA-IR c-peptide
Before
After
P value
8.8 1.1 47.4 8.3 131.2 18.1 82.1 1.9 108.0 6.75 335.9 14.8 4.4 0.3
9.6 1.3 75.4 18.4 167.0 29.7 85.8 2.6 125.1 10.9 369.0 22.5 4.8 0.4
NS .034 .06 NS NS NS NS
5.6 0.6
5.9 0.5
NS
1.1 0.1 1.6 0.2
1.2 0.2 1.7 0.2
NS NS
Note: AUC, area under the curve; GIR, glucose infusion rate; HOMA-IR, homeostasis model assessment of insulin resistance; ITT, insulin tolerance test; NS, not statistically significant; OGTT, oral glucose tolerance test. Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
Statistical Analysis The statistical analysis was performed using SigmaStat software (SPSS Inc, Chicago, IL). Statistical analysis of the results was performed using a 2-tailed Student’s t-test (paired).
mean insulin AUC after treatment (167 29.7 mU/mL) was also higher compared with before treatment (131.2 18.1 mU/mL), but the difference was not statistically significant (P¼0.06).
Data are presented as mean SE, and P<.05 was considered statistically significant.
Effects on b-cell Function The proinsulin and c-peptide levels showed no statistically significant change after treatment (Fig. 1). The HOMA-%b before (120 8.7) and after treatment (111.6 9.3) were not statistically significantly different.
RESULTS Metabolic and Endocrine Changes with Raloxifene Treatment There was no statistically significant change in the body mass index (BMI) of the participants. There was a statistically significant decrease in cholesterol (P< .03) and LDL (P< .006) levels with raloxifene treatment, but HDL and triglyceride levels did not change. The hemoglobin A1C (HbA1C) levels before and after treatment were similar (see Table 1). Effects of Raloxifene on Glucose and Insulin Levels Insulin and glucose levels before and after raloxifene treatment are shown in Table 2. Raloxifene treatment for 3 months had no statistically significant effect on fasting glucose levels (82.1 1.9 vs 85.8 2.6 mg/dL) or 2-hour glucose levels (108.0 6.7 vs 125.1 10.9 mg/dL). The glucose AUC during OGTT after treatment (369.0 22.5 mg/dL) was not statistically significantly different from before treatment (335.9 14.8 mg/dL). The fasting insulin levels after treatment (9.6 1.3 mU/mL) also were not statistically significantly different from the levels before treatment (8.8 1.1 mU/mL); however, the insulin levels at 2 hours (75.4 18.4 mU/mL) were statistically significantly higher after treatment (P<.03) compared with levels before treatment (47.4 8.3 mU/mL). The 616
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Raloxifene and insulin sensitivity
Effects on Hepatic Insulin Clearance The c-peptide to insulin molar ratio was statistically significantly decreased after treatment at 2 hours (P< .003) and the AUC (P< .007), indicating that raloxifene decreases hepatic clearance of insulin (Fig. 2). Effects on Insulin Sensitivity The HOMA-IR calculated with insulin levels (1.1 0.1) showed no statistically significant change after treatment (1.2 0.2). The HOMA-IR calculated using fasting c-peptide levels (1.6 0.2) was also not statistically significantly different (1.7 0.2) after raloxifene treatment. Insulin sensitivity, as assessed by ITT (4.4 0.3 vs 4.8 0.4), and GIR, as determined by euglycemic clamp studies (5.6 0.6 vs 5.9 0.5 mg/kg per minute), were also not significantly different after treatment compared with before treatment (see Table 2). DISCUSSION Some women are reluctant to use HT because of the potential adverse effects reported in the Women’s Health Initiative study (17). Studies in primates and humans indicate that Vol. 89, No. 3, March 2008
FIGURE 1
FIGURE 2
(A) Insulin, (B) c-peptide, and (C) proinsulin levels before and after 3 months of treatment with raloxifene. *P< .034.
C-peptide/insulin molar ratios during oral glucose tolerance test before and after 3 months of treatment with raloxifene. *P< .003, **P< .038.
Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
insulin resistance in monkeys treated with an HT consisting of conjugated estrogen and medroxyprogesterone acetate. They observed that both tibolone and HT increased insulin resistance in treated animals compared with controls. Raloxifene is a nonsteroidal benzothiofene that acts as a selective estrogen receptor modulator (SERM). Raloxifene has been approved for prevention and treatment of osteoporosis. It has estrogen-like favorable effects on bone and cholesterol metabolism, and it has antiestrogen-like effects in the uterus and breast (18). There is limited information on the effects of raloxifene on insulin resistance and glucose metabolism, and the results of previous studies have been controversial. These controversial results could be due to the methods used for the assessment of insulin resistance, duration of treatment, and characteristics of the patients studied. Although two studies indicated that raloxifene has a neutral effect on insulin sensitivity and glucose metabolism (19, 20), one study reported a worsening effect (21).
Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
progestational agents reduce insulin sensitivity and dilute the beneficial effects of estrogen (9, 10). Shadoan et al. (10) used a homeostasis model assessment (HOMA-IR) to measure Fertility and Sterility
In the present study, raloxifene had no effect on insulin sensitivity in healthy postmenopausal women. We used several accepted measures of insulin resistance to quantitate insulin resistance before and after treatment: HOMA, ITT, and euglycemic clamp studies. Furthermore, there were no changes in fasting glucose or AUC glucose levels during OGTT before or after treatment, indicating that raloxifene has no effect on glucose tolerance. These results are similar to those of Cagnacci et al. (20), who evaluated glucose tolerance and insulin sensitivity by OGTT performed before and 617
after 6 months of treatment with raloxifene. Cucinelli et al. (19) observed that raloxifene improved insulin sensitivity and decreased insulin levels in a selected population of hyperinsulinemic postmenopausal women but had no effect on normoinsulinemic postmenopausal women. All of the menopausal women who were observed in our present study had normal insulin levels before treatment. Lee et al. (21) reported that raloxifene decreased insulin sensitivity, as measured by frequently sampled intravenous glucose tolerance test in a group of older postmenopausal women (mean age: 70 years). That study was limited by the small sample size and the short duration of treatment (8 weeks). Oleksik et al. (22) observed that raloxifene use was associated with decreased insulin to glucose ratio in a group of nondiabetic postmenopausal women with osteoporosis. That all women had osteoporosis in that study may have induced a bias because of possible abnormalities in the growth hormone/insulin-like growth factor 1 (GH-IGF-I) axis in patients with osteoporosis. The insulin to glucose ratio in two fasting blood samples is not an accurate method to assess insulin resistance. Our study used several well-accepted methods (including euglycemic clamp studies, which are more sensitive) to measure insulin sensitivity. Even though there was no change in insulin sensitivity with raloxifene treatment as measured by various tests, we observed a statistically significant increase in insulin levels 2 hours after glucose ingestion and AUC during OGTT. Raloxifene does not appear to affect b-cell function, as proinsulin and c-peptide levels showed no statistically significant change with treatment. Cagnacci et al. (20) also reported that 6 months of raloxifene treatment had no affect on bcell responsiveness. In our study, the increase in postglucose insulin levels that was observed in women with raloxifene treatment was probably due to a decrease in hepatic insulin extraction rather than increased secretion. This led us to investigate the effect of raloxifene on insulin clearance by calculating the ratio of c-peptide to insulin. Insulin and c-peptide are secreted in equimolar concentrations by the pancreas, but, in contrast to insulin, very little c-peptide is removed by the liver. Thus, simultaneous measurement of c-peptide and insulin in the peripheral blood provides a noninvasive method of assessing hepatic insulin clearance (23). Even though the fasting c-peptide to insulin ratio was not different after raloxifene treatment, we did find a statistically significant decrease in the postglucose c-peptide to insulin ratio after treatment, indicating that raloxifene decreases insulin clearance after glucose ingestion. In contrast, transdermal estrogen has been shown to enhance insulin clearance (c-peptide to insulin ratio), while oral estrogen has no effect (9, 24). In the study by Cagnacci et al. (20), the c-peptide to insulin ratio was not modified by raloxifene treatment; at baseline, fasting basal insulin levels were higher and the c-peptide to insulin ratio lower in patients treated with placebo than those treated with raloxifene, which could have affected their results. Results of our study indicate that raloxifene has no adverse effect on glucose tolerance or insulin sensitivity in 618
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nondiabetic postmenopausal women. An interesting finding was the increase in 2-hour insulin levels and the insulin AUC during the OGTT after treatment with raloxifene. In the context of no changes in insulin sensitivity and b-cell function as well as the decrease in the c-peptide/insulin ratio, this finding indicates that the rise in insulin response is likely due to decreased hepatic extraction. This effect of raloxifene conserves insulin and may be beneficial in patients who have decreased insulin reserve or who are predisposed to type 2 diabetes. REFERENCES 1. Psoudler AJ, Felton CV, Stevenson JC. Aging and the response of plasma insulin, glucose and C-peptide concentrations to intravenous glucose in postmenopausal women. Clin Sci 1992;83:489–94. 2. Walton C, Godsland IF, Psoudler AJ, Wynn V, Stevenson JC. The effects of the menopause on insulin sensitivity, secretion and elimination in nonobese healthy women. Eur J Clin Invest 1993;23:466–73. 3. Poehlman ET, Toth MJ, Gardner AW. Changes in energy balance and body composition at menopause: a controlled longitudinal study. Ann Intern Med 1995;123:673–5. 4. Park YW, Zhu S, Palaniappan L, Heshka S, Carnethon MR, Heymsfield SB. The metabolic syndrome: prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey, 1988–1994. Arch Intern Med 2003;163:427–36. 5. Pyorala K, Savolainen E, Kaukola S, Haapakoski J. Plasma insulin as coronary heart disease risk factor: relationship to other risk factors and predictive value during 9 1/2 follow-up of Helsinki Policeman Study population. Acta Med Scand 1985;701:38–52. 6. Laakso M, Barret-Connor E. Asymptomatic hyperglycemia is associated with lipid and lipoprotein changes favoring atherosclerosis. Arteriosclerosis 1989;9:665–72. 7. Barret-Connor EL, Cohen BA, Wingard DL, Edelstein SL. Why diabetes mellitus a stronger risk factor for fatal ischemic heart disease in women than in men? The Rancho Bernado Study. JAMA 1991;265: 627–31. 8. Cefalu WT, Wagner JD, Bell-Farrow AD, Wang ZQ, Adams MR, Toffolo G, et al. The effects of hormonal replacement therapy on insulin sensitivity in surgically menopausal cynomolgus monkeys (Macaca fasicularis). Am J Obstet Gynecol 1994;171:440–5. 9. Lindheim SR, Presser SC, Ditkoff EC, Vijod MA, Stanczyk FZ, Lobo RA. A possible bimodal effect of estrogen on insulin sensitivity in postmenopausal women and the attenuating effect of added progestins. Fertil Steril 1993;60:664–7. 10. Shadoan MK, Anthony MS, Rankin SE, Clarkson TB, Wagner JD. Effects of tibolone and conjugated equine estrogens with or without medroxyprogesterone acetate on body composition and fasting carbohydrate measures in surgically menopausal monkeys. Metabolism 2003;52:1085–91. 11. Ettinger B, Black DM, Mitlak BH, Knickerbocker RK, Nickelson T, Genant HK, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3 year randomized clinical trial. JAMA 1999;282:637–45. 12. Gresa TA, Sluka LP, Bryant HU, Cullinan GJ, Glarebrook AL, Jones CD, et al. Molecular determinants of tissue selectivity in estrogen receptor modulators. Proc Natl Acad Sci USA 1997;94: 14105–10. 13. Andersson B, Johannsson G, Holm G, Bengtsson B, Sashegyi A, Pavo I, et al. Raloxifene did not affect insulin sensitivity or glycemic control in postmenopausal women with type 2 diabetes mellitus: a randomized clinical trial. J Clin Endocrinol Metab 2002;87:122–8. 14. Mathews DR, Hoeker JP, Rudenski AS, Nayer BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and b-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9.
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15. Rizza RA, Mandarino LJ, Gerich JE. Dose-response characteristics for effects of insulin on production and utilization of glucose in man. Am J Physiol 1981;240:E630–9. 16. Stuart CA, Nagamani M. Insulin infusion acutely augments ovarian androgen production in normal women. Fertil Steril 1990;54:788–92. 17. Cauley J, Robbins J, Chen Z, Cummings SR, Jackson RD, LaCroix AZ, et al. Effects of estrogen plus progestin on risk of fracture and bone mineral density: the Women’s Health Initiative randomized trial. Women’s Health Initiative Investigators. JAMA 2003;290:1729–38. 18. Delmas PD, Ensrud KE, Adachi JD, Harper KD, Sarkar S, Gennari C, et al. Efficacy of raloxifene on vertebral fracture risk reduction in postmenopausal women with osteoporosis: four-year results from a randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation Investigators. J Clin Endocrinol Metab 2002;87:3609–17. 19. Cucinelli F, Soranna L, Romualdi D, Muzj G, Mancuso S, Lanzone A. The effect of raloxifene on glycol-insulinemic homeostasis in healthy postmenopausal women: a randomized placebo-controlled study. J Clin Endocrinol Metab 2002;87:4186–92.
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20. Cagnacci A, Paoletti AM, Zanni A, Arangino S, Ibba G, Orru M, et al. Raloxifene does not modify insulin sensitivity and glucose metabolism in postmenopausal women. J Clin Endocrinol Metab 2002;87:4117–21. 21. Lee CC, Kasa-Vubu JZ, Supiano MA. Differential effects of raloxifene and estrogen on insulin sensitivity in postmenopausal women. J Am Geriatr Soc 2003;51:683–8. 22. Oleksik AM, Duong T, Pliester N, Asma G, Popp-Snijders C, Lips P. Effects of the selective estrogen receptor modulator, raloxifene, on the somatotropic axis and insulin-glucose homeostasis. J Clin Endocrinol Metab 2001;86:2763–8. 23. Faber OK, Christensen K, Kehlet H, Madsbad S, Binder C. Decreased insulin removal contributes to hyperinsulinemia in obesity. J Clin Endocrinol Metab 1981;53:618–21. 24. Cagnacci A, Soldani R, Carriero PL, Paoletti AM, Fioretti P, Melis GB. Effects of low doses of transdermal 17 beta-estradiol on carbohydrate metabolism in postmenopausal women. J Clin Endocrinol Metabol 1992;74:1396–400.
619
b
Department of Internal Medicine, University of Texas Medical Branch,
Objective: To investigate the effect of raloxifene on insulin sensitivity, b-cell function, hepatic insulin clearance, and glucose tolerance in postmenopausal women. Design: Prospective study. Setting: University of Texas Medical Branch at Galveston, Texas. Patient(s): Twenty normal postmenopausal women. Intervention(s): An oral glucose tolerance test (OGTT) was performed on all study participants before and after treatment with 60 mg of raloxifene daily for 3 months. Blood samples were obtained at baseline and 1, 2, and 3 hours after 75-g oral glucose administration for measurement of glucose, insulin, proinsulin, and c-peptide levels. Insulin tolerance test (ITT) and euglycemic clamp studies were also performed before and after treatment. Main Outcome Measure(s): Glucose and insulin area under curve (AUC) were calculated. The c-peptide to insulin ratio was determined to assess hepatic clearance of insulin. The homeostasis model assessment (HOMA) was used to calculate the index of insulin resistance (HOMA-IR) and b-cell function (HOMA-%b). Insulin sensitivity was assessed by insulin tolerance test and glucose infusion rate (GIR) during euglycemic clamp studies. Result(s): There was no change in fasting or AUC glucose levels. Fasting insulin levels were not statistically significantly different, but the insulin levels at 2 hours and insulin AUC were higher after treatment compared with before treatment. Proinsulin, c-peptide levels, and HOMA-%b did not change. The c-peptide to insulin molar ratio was statistically significantly decreased after treatment. There was no change in insulin sensitivity. Conclusion(s): These results indicate that raloxifene has no adverse effect on insulin sensitivity or glucose tolerance, and it does not affect b-cell function. After glucose load, raloxifene decreases hepatic insulin extraction and thus conserves insulin, which may be beneficial to patients with decreased b-cell reserve or those predisposed to type 2 diabetes. (Fertil Steril 2008;89:614–9. 2008 by American Society for Reproductive Medicine.) Key Words: Raloxifene, insulin sensitivity, b-cell function
There is a progressive increase in insulin resistance with aging and, hence, an increase in circulating insulin levels in postmenopausal women (1, 2). Poehlman et al. (3) found that postmenopausal women have increased fasting insulin levels compared with premenopausal women. Insulin resistance and associated metabolic syndrome lead to an increased risk of coronary heart disease and type 2 diabetes (4, 5). The presence of diabetes further increases the risk for fatal ischemic heart disease in women (6, 7). Previous studies of the effect of hormone therapy (HT) on insulin resistance in postmenopausal women have been controversial (8). Studies in primates and humans indicate that progestational agents reduce insulin sensitivity and dilute the beneficial effects of estrogen (9, 10). Raloxifene is a selective
estrogen receptor modulator that is approved for prevention and treatment of osteoporosis (11). It acts as estrogen receptor agonist in bone tissue, and as an estrogen antagonist in breast tissue and endometrium (12). One of the benefits of taking raloxifene instead of HT is that there is no need for additional progestational agents. Raloxifene does not affect insulin sensitivity or glycemic control in postmenopausal women with type 2 diabetes (13).
Received January 17, 2007; revised and accepted March 27, 2007. Supported in part by a grant from Eli Lilly and the National Institutes of Health GCRC M01 RR00073. Presented at the Endocrine Society Annual Meeting; San Diego, California; June 2005. Reprint requests: Manubai Nagamani, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology; University of Texas Medical Branch, Galveston, TX 77555-0587 (FAX: 281-486-5554; E-mail: [email protected]).
MATERIALS AND METHODS Patients Twenty normal postmenopausal women (45 to 62 years of age) who had their last menstrual period at least 1 year before study enrollment and had not taken any HT for at least 3 months before enrollment were included in the study. Inclusion criteria consisted of a follicle-stimulating hormone
614
The purpose of our study was to investigate the effect of treatment with raloxifene for 12 weeks on insulin sensitivity, glucose tolerance, b-cell function, and hepatic insulin clearance in normal postmenopausal women.
Fertility and Sterility Vol. 89, No. 3, March 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.03.083
(FSH) level of >40 mIU/mL and serum estradiol levels of <40 pg/mL. Women with a history of thromboembolic disease were excluded from the study. Women with any major medical illnesses, including clinically significant abnormal liver and kidney function tests, untreated thyroid disease, or diabetes mellitus, were also excluded. The study was approved by the institutional review board of the University of Texas Medical Branch (UTMB), Galveston, Texas. All participants signed an informed consent before inclusion into the study. Clinical data on the patients studied are shown in Table 1. All women maintained a 300-g carbohydrate diet for 3 days before taking an oral glucose tolerance test (OGTT). After overnight fasting, all patients were admitted to the Clinical Research Center (CRC) at UTMB. A standard OGTT, insulin tolerance test (ITT), and euglycemic clamp studies were performed on all participants. Fasting blood samples were also obtained for total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride levels. All of the women were then started on raloxifene, 60 mg daily for 12 weeks. During the last week of treatment, the patients were readmitted to the CRC, and all of the tests were repeated. Oral Glucose Tolerance Test The insulin, c-peptide, and proinsulin levels at fasting and in response to oral glucose challenge were assessed. All of the women maintained a 300-g carbohydrate diet for 3 days before the OGTT. Blood samples were obtained at fasting and at 1, 2, and 3 hours after glucose ingestion (75 g) for measurement of glucose, insulin, proinsulin, and c-peptide levels. Insulin resistance and b-cell function were assessed by homeostasis model assessment (HOMA) from fasting glucose and insulin or c-peptide concentrations (14). The HOMA values for insulin resistance (HOMA-IR) and percentage b-cell function (HOMA-%b-cell) were calculated as previously described elsewhere (14), using a HOMA computer program (HOMA 2 calculator V2.2; The University of Oxford, Diabetes Trials Unit, Headington, Oxford, UK). An ideal normal-weight individual, aged <35 years, has a HOMA-IR of 1.0 mol mU/L2 and a HOMA-%b-cell function of 100%. The c-peptide to insulin molar ratio was determined to assess hepatic insulin extraction. Euglycemic Clamp Studies Single-dose euglycemic clamp studies were performed using modifications to the method described by Rizza et al. (15). The details of our technique have been previously described elsewhere (16). Briefly, after an overnight fast, the women were maintained at bedrest until completion (approximately 6 hours). Three intravenous lines were inserted, including one for infusions, one for sampling, and one for constant withdrawal for continuous blood glucose readout using a Biostator monitor (Life Science Instruments, Miles Laboratories, Elkhart, IN). After a 90-minute baseline infusion period, four plasma samples were drawn at 10-minute intervals. At 2 hours, a primed insulin infusion of 40 mU/m2 per minute was Fertility and Sterility
TABLE 1 Metabolic and endocrine characteristics of postmenopausal women before and after raloxifene treatment. Raloxifene treatment Before
After
BMI (kg/m2) 28.6 1.5 28.6 1.4 FSH (mIU/mL) 71.8 4.6 62.2 4.6 Estradiol 14.4 2.5 13.8 2.2 (pg/mL) Triglycerides 113.5 11.4 116.6 14.3 (mg/dL) Cholesterol 198.4 7.5 185.1 5.7 (mg/dL) HDL (mg/dL) 54.4 58.1 55.9 4 LDL (mg/dL) 120.9 6.4 106.6 3.8 HbA1C 5.5 0.1 5.5 0.1
P value NS < .04 NS NS .027 NS .006 NS
Note: BMI, body mass index; FSH, follicle-stimulating hormone; HbA1C, hemoglobin A1C; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NS, not statistically significant. Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
begun and continued for 3 hours. Euglycemia (85 mg/dL) was maintained by variable infusion of 20% glucose, which was modulated based on blood glucose concentrations and continuously monitored with the Biostator monitor. During the last 30 minutes of the insulin infusion period, four plasma samples were obtained at 10-minute intervals for glucose and insulin measurements. The whole body glucose utilization at the achieved insulin level was quantitated as the mean glucose infusion rate (GIR) during the last 30 minutes of the infusion. Insulin Tolerance Test Responsiveness to exogenous insulin was evaluated by determining the rapidity of fall in plasma glucose (KITT) after an intravenous bolus of insulin of 0.1 units/kg body weight. Human insulin was diluted to a concentration of 1 unit/mL with the diluent provided by Eli Lilly (Indianapolis, IN). The plasma glucose values at 5, 10, 15, and 20 minutes were used to calculate (KITT). Hormone Assays Blood glucose was assayed by the glucose oxidase method (Beckman Glucose Analyzer, Beckman Instruments, Fullerton, CA). Insulin levels were measured by radioimmunoassay kits obtained from Diagnostic Systems Laboratories (Webster, TX). Proinsulin and c-peptide levels were measured by radioimmunoassay with kits purchased from Linco Research (St. Charles, MO). Serum triglycerides, cholesterol, and HDL levels were measured using specific enzyme methods. 615
TABLE 2 Insulin and glucose levels during oral glucose tolerance test and insulin resistance studies in postmenopausal women before and after raloxifene treatment. Raloxifene treatment OGTT Fasting insulin (mU/mL) 2-hour insulin Insulin AUC Fasting glucose (mg/dL) 2-hour glucose Glucose AUC ITT Euglycemic clamp GIR (mg/kg/min) HOMA-IR HOMA-IR insulin HOMA-IR c-peptide
Before
After
P value
8.8 1.1 47.4 8.3 131.2 18.1 82.1 1.9 108.0 6.75 335.9 14.8 4.4 0.3
9.6 1.3 75.4 18.4 167.0 29.7 85.8 2.6 125.1 10.9 369.0 22.5 4.8 0.4
NS .034 .06 NS NS NS NS
5.6 0.6
5.9 0.5
NS
1.1 0.1 1.6 0.2
1.2 0.2 1.7 0.2
NS NS
Note: AUC, area under the curve; GIR, glucose infusion rate; HOMA-IR, homeostasis model assessment of insulin resistance; ITT, insulin tolerance test; NS, not statistically significant; OGTT, oral glucose tolerance test. Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
Statistical Analysis The statistical analysis was performed using SigmaStat software (SPSS Inc, Chicago, IL). Statistical analysis of the results was performed using a 2-tailed Student’s t-test (paired).
mean insulin AUC after treatment (167 29.7 mU/mL) was also higher compared with before treatment (131.2 18.1 mU/mL), but the difference was not statistically significant (P¼0.06).
Data are presented as mean SE, and P<.05 was considered statistically significant.
Effects on b-cell Function The proinsulin and c-peptide levels showed no statistically significant change after treatment (Fig. 1). The HOMA-%b before (120 8.7) and after treatment (111.6 9.3) were not statistically significantly different.
RESULTS Metabolic and Endocrine Changes with Raloxifene Treatment There was no statistically significant change in the body mass index (BMI) of the participants. There was a statistically significant decrease in cholesterol (P< .03) and LDL (P< .006) levels with raloxifene treatment, but HDL and triglyceride levels did not change. The hemoglobin A1C (HbA1C) levels before and after treatment were similar (see Table 1). Effects of Raloxifene on Glucose and Insulin Levels Insulin and glucose levels before and after raloxifene treatment are shown in Table 2. Raloxifene treatment for 3 months had no statistically significant effect on fasting glucose levels (82.1 1.9 vs 85.8 2.6 mg/dL) or 2-hour glucose levels (108.0 6.7 vs 125.1 10.9 mg/dL). The glucose AUC during OGTT after treatment (369.0 22.5 mg/dL) was not statistically significantly different from before treatment (335.9 14.8 mg/dL). The fasting insulin levels after treatment (9.6 1.3 mU/mL) also were not statistically significantly different from the levels before treatment (8.8 1.1 mU/mL); however, the insulin levels at 2 hours (75.4 18.4 mU/mL) were statistically significantly higher after treatment (P<.03) compared with levels before treatment (47.4 8.3 mU/mL). The 616
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Effects on Hepatic Insulin Clearance The c-peptide to insulin molar ratio was statistically significantly decreased after treatment at 2 hours (P< .003) and the AUC (P< .007), indicating that raloxifene decreases hepatic clearance of insulin (Fig. 2). Effects on Insulin Sensitivity The HOMA-IR calculated with insulin levels (1.1 0.1) showed no statistically significant change after treatment (1.2 0.2). The HOMA-IR calculated using fasting c-peptide levels (1.6 0.2) was also not statistically significantly different (1.7 0.2) after raloxifene treatment. Insulin sensitivity, as assessed by ITT (4.4 0.3 vs 4.8 0.4), and GIR, as determined by euglycemic clamp studies (5.6 0.6 vs 5.9 0.5 mg/kg per minute), were also not significantly different after treatment compared with before treatment (see Table 2). DISCUSSION Some women are reluctant to use HT because of the potential adverse effects reported in the Women’s Health Initiative study (17). Studies in primates and humans indicate that Vol. 89, No. 3, March 2008
FIGURE 1
FIGURE 2
(A) Insulin, (B) c-peptide, and (C) proinsulin levels before and after 3 months of treatment with raloxifene. *P< .034.
C-peptide/insulin molar ratios during oral glucose tolerance test before and after 3 months of treatment with raloxifene. *P< .003, **P< .038.
Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
insulin resistance in monkeys treated with an HT consisting of conjugated estrogen and medroxyprogesterone acetate. They observed that both tibolone and HT increased insulin resistance in treated animals compared with controls. Raloxifene is a nonsteroidal benzothiofene that acts as a selective estrogen receptor modulator (SERM). Raloxifene has been approved for prevention and treatment of osteoporosis. It has estrogen-like favorable effects on bone and cholesterol metabolism, and it has antiestrogen-like effects in the uterus and breast (18). There is limited information on the effects of raloxifene on insulin resistance and glucose metabolism, and the results of previous studies have been controversial. These controversial results could be due to the methods used for the assessment of insulin resistance, duration of treatment, and characteristics of the patients studied. Although two studies indicated that raloxifene has a neutral effect on insulin sensitivity and glucose metabolism (19, 20), one study reported a worsening effect (21).
Nagamani. Raloxifene and insulin sensitivity. Fertil Steril 2008.
progestational agents reduce insulin sensitivity and dilute the beneficial effects of estrogen (9, 10). Shadoan et al. (10) used a homeostasis model assessment (HOMA-IR) to measure Fertility and Sterility
In the present study, raloxifene had no effect on insulin sensitivity in healthy postmenopausal women. We used several accepted measures of insulin resistance to quantitate insulin resistance before and after treatment: HOMA, ITT, and euglycemic clamp studies. Furthermore, there were no changes in fasting glucose or AUC glucose levels during OGTT before or after treatment, indicating that raloxifene has no effect on glucose tolerance. These results are similar to those of Cagnacci et al. (20), who evaluated glucose tolerance and insulin sensitivity by OGTT performed before and 617
after 6 months of treatment with raloxifene. Cucinelli et al. (19) observed that raloxifene improved insulin sensitivity and decreased insulin levels in a selected population of hyperinsulinemic postmenopausal women but had no effect on normoinsulinemic postmenopausal women. All of the menopausal women who were observed in our present study had normal insulin levels before treatment. Lee et al. (21) reported that raloxifene decreased insulin sensitivity, as measured by frequently sampled intravenous glucose tolerance test in a group of older postmenopausal women (mean age: 70 years). That study was limited by the small sample size and the short duration of treatment (8 weeks). Oleksik et al. (22) observed that raloxifene use was associated with decreased insulin to glucose ratio in a group of nondiabetic postmenopausal women with osteoporosis. That all women had osteoporosis in that study may have induced a bias because of possible abnormalities in the growth hormone/insulin-like growth factor 1 (GH-IGF-I) axis in patients with osteoporosis. The insulin to glucose ratio in two fasting blood samples is not an accurate method to assess insulin resistance. Our study used several well-accepted methods (including euglycemic clamp studies, which are more sensitive) to measure insulin sensitivity. Even though there was no change in insulin sensitivity with raloxifene treatment as measured by various tests, we observed a statistically significant increase in insulin levels 2 hours after glucose ingestion and AUC during OGTT. Raloxifene does not appear to affect b-cell function, as proinsulin and c-peptide levels showed no statistically significant change with treatment. Cagnacci et al. (20) also reported that 6 months of raloxifene treatment had no affect on bcell responsiveness. In our study, the increase in postglucose insulin levels that was observed in women with raloxifene treatment was probably due to a decrease in hepatic insulin extraction rather than increased secretion. This led us to investigate the effect of raloxifene on insulin clearance by calculating the ratio of c-peptide to insulin. Insulin and c-peptide are secreted in equimolar concentrations by the pancreas, but, in contrast to insulin, very little c-peptide is removed by the liver. Thus, simultaneous measurement of c-peptide and insulin in the peripheral blood provides a noninvasive method of assessing hepatic insulin clearance (23). Even though the fasting c-peptide to insulin ratio was not different after raloxifene treatment, we did find a statistically significant decrease in the postglucose c-peptide to insulin ratio after treatment, indicating that raloxifene decreases insulin clearance after glucose ingestion. In contrast, transdermal estrogen has been shown to enhance insulin clearance (c-peptide to insulin ratio), while oral estrogen has no effect (9, 24). In the study by Cagnacci et al. (20), the c-peptide to insulin ratio was not modified by raloxifene treatment; at baseline, fasting basal insulin levels were higher and the c-peptide to insulin ratio lower in patients treated with placebo than those treated with raloxifene, which could have affected their results. Results of our study indicate that raloxifene has no adverse effect on glucose tolerance or insulin sensitivity in 618
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nondiabetic postmenopausal women. An interesting finding was the increase in 2-hour insulin levels and the insulin AUC during the OGTT after treatment with raloxifene. In the context of no changes in insulin sensitivity and b-cell function as well as the decrease in the c-peptide/insulin ratio, this finding indicates that the rise in insulin response is likely due to decreased hepatic extraction. This effect of raloxifene conserves insulin and may be beneficial in patients who have decreased insulin reserve or who are predisposed to type 2 diabetes. REFERENCES 1. Psoudler AJ, Felton CV, Stevenson JC. Aging and the response of plasma insulin, glucose and C-peptide concentrations to intravenous glucose in postmenopausal women. Clin Sci 1992;83:489–94. 2. Walton C, Godsland IF, Psoudler AJ, Wynn V, Stevenson JC. The effects of the menopause on insulin sensitivity, secretion and elimination in nonobese healthy women. Eur J Clin Invest 1993;23:466–73. 3. Poehlman ET, Toth MJ, Gardner AW. Changes in energy balance and body composition at menopause: a controlled longitudinal study. Ann Intern Med 1995;123:673–5. 4. Park YW, Zhu S, Palaniappan L, Heshka S, Carnethon MR, Heymsfield SB. The metabolic syndrome: prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey, 1988–1994. Arch Intern Med 2003;163:427–36. 5. Pyorala K, Savolainen E, Kaukola S, Haapakoski J. Plasma insulin as coronary heart disease risk factor: relationship to other risk factors and predictive value during 9 1/2 follow-up of Helsinki Policeman Study population. Acta Med Scand 1985;701:38–52. 6. Laakso M, Barret-Connor E. Asymptomatic hyperglycemia is associated with lipid and lipoprotein changes favoring atherosclerosis. Arteriosclerosis 1989;9:665–72. 7. Barret-Connor EL, Cohen BA, Wingard DL, Edelstein SL. Why diabetes mellitus a stronger risk factor for fatal ischemic heart disease in women than in men? The Rancho Bernado Study. JAMA 1991;265: 627–31. 8. Cefalu WT, Wagner JD, Bell-Farrow AD, Wang ZQ, Adams MR, Toffolo G, et al. The effects of hormonal replacement therapy on insulin sensitivity in surgically menopausal cynomolgus monkeys (Macaca fasicularis). Am J Obstet Gynecol 1994;171:440–5. 9. Lindheim SR, Presser SC, Ditkoff EC, Vijod MA, Stanczyk FZ, Lobo RA. A possible bimodal effect of estrogen on insulin sensitivity in postmenopausal women and the attenuating effect of added progestins. Fertil Steril 1993;60:664–7. 10. Shadoan MK, Anthony MS, Rankin SE, Clarkson TB, Wagner JD. Effects of tibolone and conjugated equine estrogens with or without medroxyprogesterone acetate on body composition and fasting carbohydrate measures in surgically menopausal monkeys. Metabolism 2003;52:1085–91. 11. Ettinger B, Black DM, Mitlak BH, Knickerbocker RK, Nickelson T, Genant HK, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3 year randomized clinical trial. JAMA 1999;282:637–45. 12. Gresa TA, Sluka LP, Bryant HU, Cullinan GJ, Glarebrook AL, Jones CD, et al. Molecular determinants of tissue selectivity in estrogen receptor modulators. Proc Natl Acad Sci USA 1997;94: 14105–10. 13. Andersson B, Johannsson G, Holm G, Bengtsson B, Sashegyi A, Pavo I, et al. Raloxifene did not affect insulin sensitivity or glycemic control in postmenopausal women with type 2 diabetes mellitus: a randomized clinical trial. J Clin Endocrinol Metab 2002;87:122–8. 14. Mathews DR, Hoeker JP, Rudenski AS, Nayer BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and b-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9.
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15. Rizza RA, Mandarino LJ, Gerich JE. Dose-response characteristics for effects of insulin on production and utilization of glucose in man. Am J Physiol 1981;240:E630–9. 16. Stuart CA, Nagamani M. Insulin infusion acutely augments ovarian androgen production in normal women. Fertil Steril 1990;54:788–92. 17. Cauley J, Robbins J, Chen Z, Cummings SR, Jackson RD, LaCroix AZ, et al. Effects of estrogen plus progestin on risk of fracture and bone mineral density: the Women’s Health Initiative randomized trial. Women’s Health Initiative Investigators. JAMA 2003;290:1729–38. 18. Delmas PD, Ensrud KE, Adachi JD, Harper KD, Sarkar S, Gennari C, et al. Efficacy of raloxifene on vertebral fracture risk reduction in postmenopausal women with osteoporosis: four-year results from a randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation Investigators. J Clin Endocrinol Metab 2002;87:3609–17. 19. Cucinelli F, Soranna L, Romualdi D, Muzj G, Mancuso S, Lanzone A. The effect of raloxifene on glycol-insulinemic homeostasis in healthy postmenopausal women: a randomized placebo-controlled study. J Clin Endocrinol Metab 2002;87:4186–92.
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20. Cagnacci A, Paoletti AM, Zanni A, Arangino S, Ibba G, Orru M, et al. Raloxifene does not modify insulin sensitivity and glucose metabolism in postmenopausal women. J Clin Endocrinol Metab 2002;87:4117–21. 21. Lee CC, Kasa-Vubu JZ, Supiano MA. Differential effects of raloxifene and estrogen on insulin sensitivity in postmenopausal women. J Am Geriatr Soc 2003;51:683–8. 22. Oleksik AM, Duong T, Pliester N, Asma G, Popp-Snijders C, Lips P. Effects of the selective estrogen receptor modulator, raloxifene, on the somatotropic axis and insulin-glucose homeostasis. J Clin Endocrinol Metab 2001;86:2763–8. 23. Faber OK, Christensen K, Kehlet H, Madsbad S, Binder C. Decreased insulin removal contributes to hyperinsulinemia in obesity. J Clin Endocrinol Metab 1981;53:618–21. 24. Cagnacci A, Soldani R, Carriero PL, Paoletti AM, Fioretti P, Melis GB. Effects of low doses of transdermal 17 beta-estradiol on carbohydrate metabolism in postmenopausal women. J Clin Endocrinol Metabol 1992;74:1396–400.
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