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Insulin Sensitivity and Cardiovascular Risk Factors in Ovariectomized Monkeys with Estradiol Alone or Combined with Nomegestrol Acetate¹

Departments of Comparative Medicine (J.D.W., J.K.W., L.Z., K.A.G.), Biochemistry (M.J.T.), and Internal Medicine (J.D.W., W.T.C.), Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157-1040

Address all correspondence and requests for reprints to: Janice D. Wagner, D.V.M., Ph.D., Department of Comparative Medicine, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157-1040. E-mail: jwagner{at}cpm.bgsm.edu

	Abstract

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We have previously shown that medroxyprogesterone acetate (MPA), either alone or combined with conjugated equine estrogens (CEE), significantly decreased insulin sensitivity (SI), compared with both untreated controls and those treated with CEE alone. The purpose of this study was to determine the effects of estradiol (E₂), with and without nomegestrol acetate (NA; a potent progestin that lacks androgenic activity), on SI and arterial antioxidant activity, as determined by F₂-isoprostanes. Thirty-six adult female cynomolgus monkeys (Macaca fascicularis) were ovariectomized and fed a moderately atherogenic diet, with one of the following three treatments added to the diet, for 12 weeks: 1) no treatment (control); 2) E₂; or 3) continuous combined E₂ + NA (E₂+NA). SI and glucose effectiveness were assessed by the frequently sampled iv glucose tolerance test using a third-phase insulin infusion after 10 weeks of treatment. Cholesterol content and F₂-isoprostanes were measured in the thoracic aorta after 12 weeks of treatment. E₂ treatment resulted in a significantly greater SI, compared with control or E₂+NA-treated monkeys (10.03 ± 0.91 vs. 6.35 and 6.49 x 10^-4 min^-1 µU^-1mL; P < 0.05). In contrast to our studies of CEE and MPA, E₂+NA treatment, though reducing the SI below that of the E₂ group, did not reduce the SI below that of control monkeys. As expected, the short period of treatment resulted in no significant differences in aortic cholesterol content. There was no treatment effect on total F₂-isoprostanes (representing F₂-isoprostane formation caused primarily by autooxidation), suggesting minimal antioxidant activity. However, there was a treatment difference in the prostaglandin F₂ (PGF₂) isomer (a prostaglandin (PG) isomer formed by both autooxidation of arachidonate and cyclooxygenase activity). PGF₂ concentrations were 32% lower with E₂ treatment, compared with controls, and 36% lower, compared with E₂+NA treatment (0.48 ± 0.08 vs. 0.71 ± 0.12 and 0.75 ± 0.06; P < 0.05), suggesting differences in PG synthesis between hormone treatments. In conclusion, NA, a progestin without androgenic activity, may still affect some cardiovascular risk factors differently than estrogen-only therapy. However, it seems to be less detrimental than MPA.

	Introduction

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ESTROGEN replacement therapy (ERT) reduces the risk of coronary heart disease (CHD) by about 50% in women (1) and similarly decreases the atherosclerosis extent in nonhuman primates (2, 3). However, a progestin is typically added to the ERT regimen to reduce risk of endometrial cancer, and this may detract from estrogen’s beneficial effects. For example, we recently have described a number of studies in cynomolgus monkeys where the combined administration of conjugated equine estrogens (CEE) with medroxyprogesterone acetate (MPA) adversely affected coronary artery atherosclerosis extent (3), vascular reactivity (4), and both carbohydrate metabolism and insulin sensitivity (SI) (5, 6), compared with CEE alone.

One mechanism whereby ERT may decrease CHD risk is that of altering SI and carbohydrate metabolism. However, the effects of ERT on insulin and glucose metabolism are inconsistent, with results depending on the type, dose, length of treatment, route of administration, and procedure used to measure carbohydrate metabolism (for review, see Ref.7). Likewise, the addition of a progestin to the ERT regimen is also controversial, with many studies reporting that progestins adversely affect carbohydrate metabolism (5, 6, 8, 9, 10, 11), whereas others have not found adverse effects (12, 13, 14).

Estrogens also may decrease CHD risk via their antioxidant activity. A number of studies have found that estrogens decrease low-density lipoprotein (LDL) oxidizability (15, 16). Although estrogen’s antioxidant effects on plasma LDL may decrease CHD risk, the oxidative potential present in the artery wall may be of more relevance. In a recent study, we found that esterified estrogens decreased the amount of thiobarbituric acid-reactive substances (TBARS) present in the aorta (17). However, the TBARS assay does not necessarily reflect the extent of polyunsaturated fatty acid (PUFA) peroxidation, and TBARS are only formed from PUFA with three or more double bonds (not from linoleic acid, which is the major PUFA in lipoproteins) (18). For these reasons, in this study, we measured arterial F₂-isoprostanes. F₂-isoprostanes are products of the free radical oxidation of arachidonic acid with structures similar to those of prostaglandin (PG), particularly prostaglandin F₂ (PGF₂) (19, 20). F₂-isoprostanes are now regarded as being highly sensitive and specific markers of free radical oxidation (20).

This study was designed to examine the separate and combined effects of continuous oral estradiol (E₂) and nomegestrol acetate (NA) on SI and arterial antioxidant activity in ovariectomized cynomolgus monkeys. NA is a 19-norprogesterone derivative with potent progestin activity that lacks androgenic activity (21). Consistent with the lack of androgenicity, NA did not diminish the beneficial effects of estradiol on plasma lipid and lipoprotein concentrations in postmenopausal women (22) and did not affect glucose tolerance in premenopausal women with menstrual abnormalities (23). Thus, we hypothesized that NA would not detract from estrogen’s effects on SI and antioxidant activity.

	Materials and Methods

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Animals and experimental design

Thirty-six adult (8–12 yr of age) female cynomolgus monkeys (Macaca fascicularis) were used in this study. Monkeys were obtained from the Indonesian Primate Center (Bogor, Indonesia) and quarantined for 90 days. During the last 60 days of quarantine, the monkeys were fed a moderately atherogenic diet containing 0.28 mg cholesterol/kcal diet (2). After quarantine, the monkeys were ovariectomized, housed in groups of 4, and fed the atherogenic diet with either: 1) no hormone treatment (control, n = 12); 2) 0.1 mg E₂ added to the diet/kg BW·day (equivalent to a human oral therapeutic dose of 1.5 mg E₂/day) (E₂, n = 12); or 3) E₂ with continuous coadministration of 0.25 mg NA/kg BW·day (equivalent to 1.5 mg E₂ and 3.75 mg NA per day) (E₂+NA, n = 12). Monkey equivalents were calculated by comparing the average number of calories consumed by people (1800 kcal/day) vs. monkeys (120 kcal/kg·day) to account for differences in body weight and metabolism between species. The study period was 12 weeks, at which time the monkeys were necropsied and arterial samples collected. All procedures involving animals were conducted in compliance with state and federal laws, standards of the Department of Health and Human Services, and guidelines established by the Institutional Animal Care and Use Committee.

Clinical chemistry analyses

Blood samples for measurement of plasma E₂ concentrations were collected 4 weeks after starting the experimental diet and just before necropsy. Reported values are the mean of both samplings. NA samples were collected just before necropsy. Samples were collected 4 h after feeding, thus representing peak hormone levels. Plasma E₂ concentrations were determined by RIA (5), and NA concentrations were determined by enzyme immunoassay after specific extraction (24).

Blood samples for measurement of plasma lipids and lipoproteins were obtained at baseline and at the end of the experiment. Baseline plasma total cholesterol and high-density lipoprotein cholesterol (HDL-C) were determined using enzymatic methods previously described (17). Posttreatment measurement of plasma lipoprotein concentrations and LDL particle size were determined by nuclear magnetic resonance spectroscopy (25).

Fasting plasma glucose and insulin measures were determined at baseline (after ovariectomy, while consuming the atherogenic diet) and after 10 weeks of treatment (at time 0 of the minimal model studies). As described previously, glucose determinations were made using the glucose oxidase method on a Glucose Analyzer 2 (Beckman Instruments, Brea, CA), and insulin was assayed by RIA (Incstar, Stillwater, MN) (6). Plasma insulin and glucose measurements were determined in duplicate. Body weights are reported at baseline and at the time of minimal model analysis.

Minimal model analysis

SI and glucose effectiveness were assessed by the frequently sampled iv glucose tolerance test using a third-phase insulin infusion (modified minimal model) (26, 27) after 10 weeks of treatment. After an overnight fast, monkeys were anesthetized with ketamine hydrochloride (15 mg/kg, im) and weighed, and an indwelling catheter was placed in the saphenous vein. One-milliliter baseline blood samples were collected into tubes containing fluoride at -5 and 0 min, and glucose (0.5 g/kg) was injected over 30 sec at time 0. The catheter was thoroughly flushed and 1-mL blood samples were taken at minutes 2, 3, 4, 5, 8, 10, 12, 14, 16, 18, and 20. After the 20-min sample, insulin (0.15 U/kg) was injected and blood collected at 22, 24, 28, 32, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, and 180 min. Plasma glucose and insulin concentrations were determined as described above.

Arterial assessments

Arterial cholesterol and isoprostane measurements were determined in sections of thoracic aorta that were frozen in liquid nitrogen and stored frozen at -70°C until analysis. Aortic lipid extracts were prepared using the method of Folch et al. (28). Total and free cholesterol content were determined enzymatically (17). Cholesteryl ester content was calculated as the difference between total and free cholesterol. Isoprostanes were measured using negative ion-selective ion-monitoring gas chromatography-mass spectroscopy as reported by Morrow et al. (19). Residual adventitia was removed after soaking the artery in 0.1 mmol/L diethylenetriamine pentaacetic acid to scavenge transition metals, and 80 µm butylated hydroxytoluene to inhibit further oxidation. Tissues were ground in liquid nitrogen and then extracted using the method of Folch et al. (28), including 0.1 mmol/L diethylenetriamine pentaacetic acid, 80 µm butylated hydroxytoluene, and 2 µm triphenylphosphine in the extraction to prevent further oxidation. After separating the aqueous layer, the protein was pelleted by low-speed centrifugation, digested in 1.7 N NaOH (2 days), and quantified by the method of Lowry et al. (29) using a BSA standard digested in the same fashion. The organic layer was dried down in vacuo and then taken up in methylene chloride-methanol with d₄-PG F₂ (Cayman Chemical Co., Ann Arbor, MI) added as an internal standard. The neutralized sample was loaded on a preconditioned C-18 cartridge, and the prostanoid fraction was eluted. After drying, the sample was taken up in ethyl acetate-hexane, applied to a silica cartridge, and the prostanoids were eluted with ethylacetate-ethanol. The eluant was dried, converted into pentafluorobenzyl esters, and then separated from less polar impurities by thin-layer chromatography using ethanol-hexane. The ester band was eluted with methanol, dried down, converted to trimethylsiloxyl ethers with bis(trimethylsilyl)trifluoroacetamide in pyridine, and then analyzed on a Ribermag R10–10C quadrupole mass analyzer (Nermag, SA, France) interfaced to a Hewlett-Packard model 5890 Series II gas chromatograph (Hewlett Packard, Wilmington, DE). The 30 m x 0.25 mm DB Wax (J & W Scientific, Rancho Cardova, CA) column introduced samples directly into the mass spectrometer source. The injection temperature was set at 270°C, and the transfer line was set at 250°C. The oven program-ramp temperature was set from 100°C to 235°C at 25°C/min, held for 8 min, then ramped to 250°C and held for an additional 5 min. Data acquisition and reduction used Vector II software by Teknivent.

Data analysis

Data are presented as mean ± SEM. ANOVA or covariance (when baseline measures were available) was used to determine treatment effects. SI and glucose effectiveness were covaried by baseline insulin:glucose ratio, because they were significant (P = 0.01) pretreatment predictors. If there was a significant treatment effect, pairwise comparisons were made. Pearson product-moment correlations were used to assess the relationship among variables. Differences were considered statistically significant at P 0.05. Two monkeys in the E₂ group did not eat the diet and were removed from the study. An insufficient number of blood samples was collected for one monkey (E₂ group) in the minimal model analysis, and thus, SI and glucose effectiveness could not be calculated for that animal.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
As expected, E₂ concentrations were significantly increased with E₂ and E₂+NA treatment, and NA concentrations were significantly increased in monkeys treated with the combination, compared with controls or E₂ treatment alone (Table 1). There were no significant differences in plasma concentrations of total cholesterol and HDL-C at baseline or during treatment. LDL particle size tended to be smaller in hormone-treated monkeys (P = 0.06). Although the E₂-treated monkeys were slightly heavier at baseline, with treatment these monkeys weighed less than those in the other groups (P = 0.15).

View larger version (21K): [in this window] [in a new window]   Figure 1. Minimal model analyses providing measures of SI and glucose effectiveness (SG) for control monkeys (open bars), those treated with E2 (hatched bars), or those treated with E2 plus NA (closed bars). Bars with unlike letters represent significant treatment differences.

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View larger version (30K): [in this window] [in a new window]   Figure 2. Arterial PGF2 concentrations for control monkeys (open bars), those treated with E2 (hatched bars), or those treated with E2 plus NA (closed bars). Bars with unlike letters represent significant treatment differences.

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	Discussion

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Consistent with the improvement in SI, E₂-treated monkeys weighed slightly less than control or combination-treated monkeys (Table 1). In studies of longer duration, we have found that E₂ treatment results in significantly lower body weights and less abdominal body fat content, as determined by computed tomography (27). In women, hormone treatment is also associated with lower body weight and body fat (12, 14, 30) and may explain some of the improvements in carbohydrate metabolism and CHD risk. In contrast to estrogens, progestins tend to increase body weight and body fat (5), and treatment with NA alone in women has been shown to increase body weight slightly (23). Fasting glucose concentrations were slightly (but significantly) higher with E₂ treatment, and glucose effectiveness tended to be lower with E₂ administration. Similarly, in the Postmenopausal Estrogen/Progestin Interventions Trial (14), although a slight decrease in fasting glucose and insulin was found with hormone treatments (primarily CEE), an increase in the 2-h glucose levels (after an oral glucose tolerance test) was found, compared with the placebo group. Godsland et al. (10) also reported a deterioration in glucose tolerance and suggested that it was caused by the decreased plasma insulin levels with estrogen. Despite the higher fasting glucose concentrations found in the present study, insulin:glucose ratios were slightly lower with E₂ treatment, consistent with the greater SI, as determined by the minimal model analyses. There were no significant differences in plasma total or HDL-C concentrations among treatment groups, but average LDL particle size was slightly lower in both hormone-treated groups, consistent with previous studies of estrogen treatment (17, 27).

Previous studies of hormonal effects on carbohydrate metabolism have yielded conflicting results. Variations caused by testing procedures used to monitor carbohydrate metabolism and the type, dose, length of time on therapy, and route of administration of hormone therapy are all important variables in insulin and glucose responses to therapy. For example, in a series of studies by Lobo’s group, using an insulin tolerance test to determine SI, the effect of oral CEE was dose dependent, i.e. a high dose (1.25 mg/day) resulted in decreased SI, and a lower dose (0.625 mg/day) resulted in increased SI (8). However, the addition of MPA (10 mg/day) to CEE (0.625 mg/day) attenuated the beneficial effects of CEE on SI (8), whereas lower doses of MPA (2.5 mg/day) did not affect SI (31). Although the high dose of oral CEE decreased SI, both a low dose (0.05 mg/day) and a high dose (1 mg/day) of transdermal E₂ (9) improved SI, suggesting that the first-pass effect may explain the adverse effect of the higher oral dose. As with oral CEE, the addition of MPA (10 mg/day) to the transdermal E₂ regimen resulted in reduced SI (9). Adverse effects of progestins on SI have been reported in other studies in women (10, 32) and in our previous studies in cynomolgus monkeys (5, 6). Furthermore, both SI (33) and insulin receptor concentrations (34) have been shown to decrease during the luteal phase of the menstrual cycle (when progesterone levels are higher), suggesting that even endogenous progesterone in normal, cycling women detracts from E₂’s beneficial effect. Although a previous study, using NA in premenopausal women, found no adverse effects on carbohydrate metabolism, these women had menstrual abnormalities, and may not have had normal follicular-phase E₂ concentrations, and thus, may have already had impaired carbohydrate metabolism (23).

The adverse effects of progesterone and progestins on SI may be caused by a number of factors. Progestins have varying amounts of androgenic activity and may even have glucocorticoid activity, which could adversely affect carbohydrate metabolism (21, 35, 36). For instance, although both are synthetic progestins, MPA was reported to be androgenic (35), whereas NA was not (21). Also, in contrast to other progestins, NA does not inhibit adrenocortical function or affect sodium balance, as do progesterone and MPA (21). Other mechanisms may involve decreased insulin clearance (9) and effects on insulin receptor number (34) with progestins.

An additional mechanism, whereby estrogens may decrease CHD risk, involves antioxidant activity. Estrogens have been shown to increase the resistance of LDL to oxidation (15, 16, 37). However, the antioxidant effects within the arterial wall may more accurately reflect cardiovascular risk. Thus, we used a gas chromatography-mass spectroscopy technique to measure arterial F₂-isoprostanes as an index of lipid peroxidation. Typically, there is a constant ratio between total F₂-isoprostanes and the PGF₂ isomer. However, in this study, the ratio of the peak correlating with PGF₂ was not constant, leading us to calculate PGF₂ separately from the other isomers. In doing so, we found that, despite no treatment effect on total F₂-isoprostanes, E₂-treated animals had 32% lower PGF₂ concentrations (compared with controls) and 36% lower (compared with combined E₂+NA treatment) (P < 0.05; Fig. 2). In a previous study (17), we found that esterified estrogens decreased the arterial content of TBARS by about 50%, compared with ovariectomized controls. Whether the greater percent reduction in products of lipid peroxidation with esterified estrogens in the previous study is caused by different methodologies, different types of estrogens, or both is unknown. However, there have been a number of reports showing varying antioxidant activity among different estrogenic steroids (15, 37).

F₂-isoprostanes have structures similar to PGs that are generated enzymatically from arachidonic acid and, a small fraction of the isoprostanes are identical to the enzymatically generated prostanoids (19, 38). Therefore, for example, the measurement of PGF₂ includes both PGF₂ generated by autooxidation and that formed by a cyclooxygenase-dependent pathway. The isoprostane products are formed at a constant ratio under specified conditions. Measured increases in the natural PGs, in this case PGF₂, suggest that a natural, physiologic response has been activated to cause the cyclooxygenase-dependent formation of PGF₂.

Sex hormones have been found to affect a number of pathways in PG formation. For example, estrogens have been shown to increase prostacyclin formation in human umbilical vessels (39, 40), and cyclooxygenase and prostacyclin synthase activities in rat aortic smooth-muscle cells (41). In contrast, estrogens have little effect on thromboxane synthesis (40) or result in decreased thromboxane formation (42). Although fewer studies have addressed PGF₂ levels in vascular cells, rats in the proestrus stage (i.e. with high E₂ concentrations) had less aortic PGF₂ production than ovariectomized rats and less contractile response to norepinephrine (43). Progesterone has been shown to increase PGE₂-9-ketoreductase activity, which converts PGE₂ to PGF₂ in follicles (44). Whether this increase also occurs in cells of the artery wall is unknown. However, it is possible that with estrogen treatment, there is a shunting of arachidonic acid and PG intermediates to prostacyclin formation, resulting in vasodilation; whereas with progestins or no hormone treatment, there may be decreased prostacyclin and increased PGF₂ formation, which could result in vasoconstriction. However, in parallel studies done using the same animals, the beneficial effects of E₂ on coronary artery vascular reactivity remained unchanged with combined E₂+NA (Williams JK et al., unpublished data).

The present study was a short-term study and was not designed to permit development of atherosclerotic lesions. Thus, the lack of treatment differences in arterial cholesterol content (Table 3) was not unexpected, and it was consistent with previous ERT studies (also of 12 weeks’ duration) (45). However, longer duration of ERT does result in decreased progression of atherosclerosis in monkeys (3, 27) and is consistent with decreased CHD risk in women (1). The limited variation in arterial cholesterol content in this study may have impaired our ability to determine whether treatment changes in SI or isoprostane formation resulted in differences in lesion extent. Regardless, there were the expected positive correlations between plasma cholesterol and LDL size with cholesterol content. Interestingly, there was a negative correlation between PGF₂ and aortic cholesterol content, whereas there was no correlation with SI and cholesterol content.

In conclusion, oral E₂ treatment in ovariectomized monkeys resulted in greater SI and less PGF₂ formation, compared with control animals, which may represent additional non-lipid-mediated mechanisms, whereby ERT improves CHD risk. The beneficial effects of E₂ on these parameters were attenuated by the addition of NA. However, the addition of NA did not reduce SI below that of control monkeys. Because increases in progesterone concentrations during the luteal phase also decrease SI, it is not surprising that even a progestin without androgenic activity, like NA, may reduce the SI level to that of control animals. Also, this study used a continuous combined estrogen-progestin therapy, which may be more detrimental than sequential progestin therapy.

	Acknowledgments

 
The authors gratefully acknowledge Ms. Karen Klein for editorial comments, and Jamie Fox and Vickie Hardy for technical assistance.

	Footnotes

 
¹ This work was supported, in part, by a grant from Laboratoires Theramex, Monaco.

Received May 29, 1997.

Revised September 18, 1997.

Accepted November 24, 1997.

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