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Original Studies |
Wisconsin Regional Primate Research Center (J.R.E., D.A.D., J.W.K., D.H.A.), Department of Obstetrics and Gynecology (J.R.E., D.H.A.), Endocrinology-Reproductive Physiology Program (J.R.E., D.H.A.), and Department of Physiology (J.W.K.), University of Wisconsin, Madison, Wisconsin 53715-1299; and Department of Obstetrics and Gynecology, Mayo Clinic (D.A.D.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Prof. David H. Abbott, Department of Obstetrics and Gynecology, Wisconsin Regional Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, Wisconsin 53715. E-mail: abbott{at}primate.wisc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to androgen-induced changes in the hypothalamo-pituitary-gonadal axis, anovulatory prenatally androgenized monkeys show insulin resistance from obesity (4), suggesting the functional integration of insulin-glucose homeostasis and ovarian function in primates. This hypothesis is further supported by the finding that women with polycystic ovarian syndrome (PCOS) exhibit a constellation of disorders characterized by LH hypersecretion (5), hyperandrogenic anovulation (6), and reduced peripheral insulin sensitivity (SI), leading to glucose intolerance in 20–30% of such individuals (7).
To date, however, the degree to which prenatal androgen exposure in adult female rhesus monkeys permanently alters insulin-glucose homeostasis remains unknown. Therefore, the present study examined SI and pancreatic ß-cell function in prenatally androgenized and normal adult female rhesus monkeys undergoing iv glucose tolerance test (FSIGT). The study demonstrated that prenatal androgen excess perturbed insulin-glucose homeodynamics regardless of gestational age. Early gestational androgen excess may impair insulin secretion by pancreatic ß-cells, whereas late gestational androgen alters insulin sensitivity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 34 adult female rhesus monkeys (Macaca mulatta) used in this study were maintained at the Wisconsin Regional Primate Research Center (WRPRC) according to standard protocol (1, 8). Animals were fed Purina monkey chow (Ralston Purina Co., St. Louis, MO; product no. 5038) with occasional supplementation of fresh fruits and bread. This formulation of monkey chow provides 70% of calories as carbohydrate, 13% as fat, and 17% as protein. The Graduate School animal care and use committee of the University of Wisconsin–Madison approved all experiments and animal protocols. Animal maintenance was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act with its subsequent amendments. Prenatally androgenized females were developed as previously reported (1). Briefly, 19 prenatally androgenized females were produced by injecting pregnant rhesus monkeys carrying female fetuses with 10 mg testosterone propionate (TP), sc, for 15–35 consecutive days. The TP injections were initiated either on gestational day 40 (early treated; n = 10) or between days 100–115 (late treated; n = 9; total gestation, 165 days). The early treated, prenatally androgenized females had external genital masculinization and obliteration of the external vaginal orifice, whereas the late treated, prenatally androgenized females showed no genital virilization except for clitoromegally. All prenatally androgenized females displayed masculinized behavior independent of genital masculinization (9). The control group for the study consisted of 15 females that were not exposed to prenatal androgen treatment. The early treated and late treated, prenatally androgenized females as well as the control females were of similar midreproductive ages (15.5 ± 0.8, 15.4 ± 0.9, and 16.9 ± 0.9 yr), body weights (8.4 ± 0.7, 8.2 ± 0.2, and 8.5 ± 0.5 kg), and body mass indexes [BMIs; body weight (kilograms)/crown-rump length (meters squared); 37.4 ± 2.6, 35.9 ± 1.8, and 37.5 ± 1.7 kg/m2, respectively].
Experimental procedures
Determination of ovulatory status. To determine ovulatory status, each female underwent daily perineal inspection for the presence of menses and twice weekly blood sampling (between 0800–1000 h) for serum progesterone determinations. Each animal was studied for 90 days (approximately three menstrual cycles). A menstrual cycle was considered ovulatory if two serum progesterone values greater than 1 ng/mL were obtained 15 days before menses (1, 3). Experimental procedures were performed within 5 days of menses in ovulatory females (e.g. early follicular phase) or at random in anovulatory females. All procedures were performed between the months of September and May to avoid possible seasonal quiescence in menstrual activity (10).
FSIGT. Each animal underwent a single FSIGT as previously described (12). Briefly, after an overnight fast, each animal was anesthetized with ketamine hydrochloride (15 mg/kg, im) and diazepam (1.25 mg/kg, im). Supplemental ketamine was administered as appropriate to maintain anesthesia (5–10 mg/kg, im). A catheter was placed into the vena cava through the saphenous vein for blood sampling (e.g. 32 samples over 195 min) and for administration of glucose (300 mg/kg at 0 min) and tolbutamide (5 mg/kg at 20 min).
Assay procedures. All assays were performed at the WRPRC Assay Services Laboratory as previously described (11, 12). Glucose was measured by the glucose oxidase method [intra- and interassay coefficients of variation (CVs), respectively, 2.9% and 4.0%]. Insulin was determined by RIA (CVs, 2.8% and 6.9%). Progesterone was measured by enzyme immunoassay (CVs, 4.6% and 12.9%).
Data analysis and statistical methods
Summary measures derived from FSIGT. Insulin sensitivity (the measure of the fraction of glucose cleared from the circulation per unit increase in insulin) and glucose effectiveness (Sg; the measure of the ability of glucose to increase its own uptake and to suppress hepatic glucose production at basal insulin levels) were determined using the modified minimal model method (13). Further measures derived from the FSIGT were basal insulin (Ib; mean of the four prechallenge plasma insulin values, -15, 10, -5, and -2 min), basal glucose (Gb; average of the four prechallenge plasma glucose values), acute insulin responses to glucose (AIRg; average elevation of posthepatic plasma insulin concentration above the baseline for the 2, 3, and 4 min samples), acute insulin response to tolbutamide (AIRtol; average elevation of posthepatic plasma insulin concentration above the baseline for the 22, 23, and 24 min samples), glucose disappearance rate (KG; slope of the log linear regression of plasma glucose concentration between 10 and 19 min), and disposition index (DI or ß-cell compensation index; product of SI and AIRg) (12).
Percentile ranking for the hyperbolic relationship between
SI and AIRg. The hyperbolic relationship
between SI, as the independent variable and AIRg
as the dependent variable was determined using data obtained from 30
normal adult female rhesus monkeys fed a special, purified diet,
undergoing FSIGT (age, 11.3 ± 0.4 yr; BW, 7.2 ± 0.3 kg;
BMI, 31.1 ± 1.1 kg/m2) (14, 15) by
previously described human methodology (16). Log transformation of
SI and AIRg (r2 = 0.45;
P 
0.0001; Fig. 1
)
created the best-fit hyperbolic line compared to a linear fit line
(r2 = 0.21; P 0.01). Standard
least squares regression, which only accounts for error in the
dependent variable, was used because the range of error for
SI was relatively small compared to the range of
values for the dependent variable (17). The hyperbolic relationship
between SI and AIRg (Z
= ([ln
(AIRg) - 6.17 + 0.67 x
ln(SI)])/0.594) was derived from the equation
for the hyperbolic curve. The Z score for each study animal was
calculated using their SI and AIRg values, with
the corresponding percentile ranking determined from a standard table
of normal distribution.
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Glucose and insulin values were submitted to log transformation,
when appropriate, to achieve homogeneity of variance and to increase
linearity (18). Normality of variables was confirmed using a Lilliefors
test (two-tailed). All variables were compared by nested ANOVA, using
female group (control vs. prenatally androgenized female)
and gestational timing of TP exposure (early vs. late
treated, nested within prenatally androgenized female) as factors to
determine the independent effects of these variables and possible
interactions (Systat Macintosh version 5.2, Systat, Evanston, IL). When
significant statistical interactions were present by ANOVA,
post-hoc t tests (two-tailed) were performed on the
variables. Regression analyses were performed using BMI as the
dependent variable and data obtained from the minimal model as
independent variables. P 0.05 was considered
significant. Minimal model data were expressed as antilog of the
transformed means and 95% confidence limits, whereas percentile
rankings and BMI were expressed as the mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DI was decreased in the group of early treated, prenatally
androgenized females compared to that in control females (P
0.05; Table 1). Conversely
SI and DI were significantly greater in the group
of late treated, prenatally androgenized females than in early treated,
prenatally androgenized and control females (SI,
P 0.05; DI, P 0.03). The
SG, Ib,
Gb, AIRg, AIRtol, and KG
were similar in both groups of prenatally androgenized females as well
as in the group of control females.
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Individual percentile rankings for the hyperbolic relationship
between SI vs. AIRg for all early
treated, prenatally androgenized females were below the best-fit line
(e.g. the 50th percentile; Fig. 2). The individual
percentile rankings for SI vs. AIRg
for late treated and control females were more evenly distributed
around the 50th percentile. Consequently, the mean percentile ranking
for the SI vs. AIRg hyperbolic curve
in the group of early treated prenatally androgenized females was
decreased compared to those in the late treated, prenatally
androgenized female and control female groups (10.3 ± 3.4
vs. 40.5 ± 9.5 and 31.3 ± 5.8; P
0.01).
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There was no statistical relationship between
SI and BMI in the groups of early treated,
prenatally androgenized and control females given the range of BMI for
these animals. In contrast, there was a negative relationship
(r2 = 0.5; P 0.03) between
SI and BMI in the group of late treated,
prenatally androgenized females. There were no statistical
relationships between BMI and Sg,
Ib, Gb, AIRg, AIRtol,
KG, or DI in any female group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early gestational androgen excess in female rhesus monkeys appears to impair pancreatic ß-cell function. The DI, a relative index of the relationship between acute pancreatic insulin responsiveness (e.g. AIRg) and the ability of insulin to induce glucose uptake (e.g. SI), was decreased in early treated, prenatally androgenized females, indicating a diminished ability of pancreatic ß-cells to respond to hyperglycemic episodes (19, 20). The mean percentile ranking for the hyperbolic relationship between SI and AIRg in early treated, prenatally androgenized females was also significantly reduced. In humans, percentile rankings for the same relationship (e.g. SI and AIRg) below the 50th percentile (the median percentile for a healthy, normative population) indicate diminished pancreatic ß-cell secretion of insulin and occur when reduced SI values are combined with low or normal values of AIRg (16, 19). We found evidence of such diminished pancreatic function in early treated, prenatally androgenized females alone. All of these females had percentile rankings below the 50th percentile, ranking below age- and body size-matched controls regardless of degree of adiposity.
As androgen receptors have been identified in the primate endocrine pancreas (21), excess androgen during early prenatal life may permanently alter pancreatic development in a manner similar to that described in tissues of the central nervous system (e.g. neural centers regulating sexual behavior) and reproductive tract (22). Androgen excess in the early treated female rhesus monkeys coincides with pancreatic organogenesis (23) and therefore could have adversely affected differentiation of the pancreas, leading to diminished pancreatic ß-cell function. Moreover, if androgen excess at this early stage of development also decreases hepatic insulin clearance in monkeys, as it does in rats (24), our experimental findings may have underestimated the differences found in insulin-glucose homeodynamics between early treated, prenatally androgenized females and the other two groups of females.
Late gestation androgen excess was associated with a negative relationship between SI and adiposity that was not found in early treated or control females. Prenatal androgen excess during late gestation may result in a unique alteration in body composition, where total body adiposity is decreased with a relative increase in the proportion of fat in the viscera. This assumption would agree with studies of perinatally androgenized female rats, which exhibited decreased total body fat in combination with increased proportion of visceral fat (25). Additionally, perinatal androgen excess in female rats is deleterious to hepatic insulin action by diminishing hepatic insulin binding and receptor function (24) which would be exacerbated by the increasing proportion of fat in the viscera. Such body composition changes are also demonstrated with long term androgen exposure in female to male human transsexuals with similar decreases in total body fat and increases in visceral fat (26). Therefore, in late treated, prenatally androgenized females, although insulin sensitivity may be increased in the lean individuals, a propensity for visceral adiposity as body mass increases may excessively promote insulin resistance and thus the negative relationship between BMI and SI.
In the present study, the effects of estrogen on insulin-glucose homeodynamics were controlled by performing all FSIGTs during the early follicular phase of the menstrual cycle or during periods of anovulation. Estrogen levels in anovulatory prenatally androgenized females do not differ from early follicular phase values in ovulatory prenatally androgenized and control females (4). Estrogen plays an important role in modulating peripheral and hepatic insulin sensitivities. Elevated levels of circulating estrogen are associated with increased SI in women and female rhesus monkeys alike (14, 27), and in vitro treatment of rat hepatocytes with estradiol increases insulin binding and receptor-mediated insulin clearance (24).
It is theoretically possible that prenatal androgen exposure in women can induce a PCOS-like condition without causing external genital virilization. As the human fetal ovary is capable of steroidogenesis during the second trimester of intrauterine life, abnormal ovarian androgen production could occur during a time of human development when first trimester external genital differentiation is complete (28, 29). This hypothesis is supported by the findings in diabetic pregnancies of elevated amniotic fluid androgen levels of presumed fetal ovarian origin and ovarian thecal-lutein cell hyperplasia in the subsequent female infants (30, 31). Furthermore, the fetal adrenal is steroidogenically active during midtrimester human development (32) and might additionally contribute to in utero hyperandrogenism. For example, congenital adrenal virilizing cancer has been associated with hirsutism, ovarian hyperandrogenism, and LH hypersecretion in an adolescent girl without genital ambiguity (33). Although the effect of prenatal androgen exposure in women on glucose-insulin regulation is unclear, congenital rubella syndrome recently has been linked to both PCOS and diabetes, but not genital ambiguity (34), thereby demonstrating that perturbation of normal human fetal development can induce subtle, yet permanent, abnormalities of both reproductive and metabolic function.
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Acknowledgments |
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We thank S. G. Eisele, K. M. Boehm, and the Animal Care Staff of WRPRC for management and maintenance of the animals; C. O’Rourke for veterinary care, G. R. Scheffler, T. E. Ziegler, D. J. Wittwer, F. H. Wegner, and S. T. Baum for assistance with hormone assays and glucose; M. K. Clayton for assistance with statistical analysis; and R. J. Colman for technical assistance.
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Footnotes |
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Received April 16, 1999.
Revised November 19, 1999.
Accepted November 30, 1999.
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References |
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