Effects of estradiol and progesterone on diabetes-associated utero-ovarian atrophy in C57BLKsJ dbdb mutant mice.код для вставкиСкачать
THE ANATOMICAL RECORD 225:310-317 (1989) Effects of Estradiol and Progesterone on Diabetes-Associated Utero-Ovarian Atrophy in C57BL/KsJ (db/db) Mutant Mice DAVID R. GARRIS Department of AnatomylDivision of Basic Sciences, Cleveland Research Laboratory, Kansas City, Missouri 64131 ABSTRACT The modulating effects of estradiol (E: 1 pg13.5 days) and progesterone (P: 2 mgl3.5 days) on the obesity and hyperinsulinemic and hyperglycemic components of the diabetes-obesity syndrome in female C57BLlKsJ (dbldb) mice, which includes cellular atrophy and adiposity in the reproductive tract, were examined and compared to corresponding control (+ /?) parameters. All control and diabetic mice received oil (vehicle control), E, or P treatments starting at 4 weeks of age. Body weight, serum insulin levels, blood glucose concentrations, and uteroovarian lipoprotein lipase activites were analyzed at 8 and 16 weeks of age and related to the ultrastructural changes in the steriod-sensitive uterine epithelium during the treatment period. Neither E nor P had any effect on body weights in (+ l?) or (dbldb) mice. The pronounced diabetes-associated elevation in serum insulin levels was enhanced by E, and suppressed by P, in 16-week-old(dbldb) mice as compared with controls. By 16 weeks of age, the E therapy normalized blood glucose levels in diabetic mice to control levels, whereas P was ineffective in modulating the hyperglycemia. The reduction in blood glucose levels in E-treated diabetic mice correlated temporally with the return of normal intracellular structure including the disappearance of intracellular lipid vacuoles characteristic of uterine epithelium cells of (dbldb) mice. The diabetes-induced rise in utero-ovarian lipoprotein lipase activity was normalized by P-therapy. The reduction in uteroovarian lipoprotein lipase activity coincided temporally with the demonstrated intracellular reorganization in (dbldb) reproductive tract tissues. These data indicate that E and P therapies are effective modulators of mutation-induced structural, metabolic, and functional changes in the reproductive tract and peripheral tissues of genetically diabetic C57BLlKsJ (dbldb) mice. The ability to restore affected tissue structure and function in this mutant mouse model to that of controls suggests that gonadal steroids may correct, delay, prevent, or suppress the diabetes-associated tissue atrophy and adiposity which characterizes this strain. Recent studies have demonstrated that the expressed diabetes-obesity syndrome in C57BLlKsJ (dbldb) mice is accompanied by adiposity and atrophy of the female reproductive tract (Garris, 1985; Garris et al., 1985a-q 1986).Alterations in follicular growth and recruitment patterns, ovarian steroid production, corpus luteum formation, cellular and interstitial structure, uterine epithelial activity, endometrial sensitivity to exogenous steroid therapy, and ovarian refractoriness to pituitary gonadotrophin stimulation are well-recognized consequences of the diabetes-obesity syndrome in this mutant mouse model (Barlett and Garris, 1987; Coleman, 1967; Garris, 1985, 1987b; Johnson and Sidman, 1979; Garris et al., 1984a,b, 1985a-c, 1986). In addition, the accompanying changes in pancreatic, hepatic, renal, neuronal, and peripheral nerve structure and function are associated with the expression of the altered carbohydrate metabolism and imbalanced glucose homeostasis, characterized by overt hyperglycemia and hyperinsulinemia [Bower et al., 1980; Bray 0 1989 ALAN R. LISS, INC. and York, 1979; Coleman, 1967, 1978, 1982; Garris et al., 1985a-c, Gartner, 1979; Hanker et al., 1980; Like et al., 1974; Meade et al., 1981;Moore et al., 1980; Sima and Robertson, 1979). Changes in cellular glucose uptake rates (Garris et al., 1984a,b, 1985a-c; Garris and Michel, 1988), as well as noradrenergic counterregulatory influences (Garris, 1988b1, exacerbate the diabetes-associated depression in intercellular processing, responsivity , sensitivity, structure, and function (Garris, 1985; Garris et al., 1985a-q 1986). As such, the impaired homeostasis stimulates enhanced cellular lipid anabolism and increased glucose and protein catabolism (Garris et al., 1985a<), resulting in a type I1 diabetes-obesity syndrome. Both steroidal and opiate hormones, as well as prehormone agents, modify the severity of the genetically Received February 14, 1989; accepted April 13, 1989. 311 ESTRADIOL, PROGESTERONE, AND DIABETES TABLE 1. Age- and diabetes-related changes in body weight, blood glucose concentrations, and serum insulin levels in control (+ /?) and diabetic (db/db)C57BL/KsJ mice: effects of estradiol and progesterone therapy' Age 8 8 8 8 16 16 16 16 Genotype ( N Treatment OilIElP Oil E + I?) (dbldb) ( P + I?) OilIEIP Oil (dbldb) E P Body weight (gm) 21.8 t 0.3 42.1 t 1.4* 43.2 t 2.8" 42.8 t 1.8* 24.5 t 0.6 56.0 t 3.9* 66.7 t 1.5* 52.9 t 6.8* Blood glucose(mg/dl) 136 t 4 308 t 22* 251 t 9* 255 t 25* 142 t 3 341 t 16* 163 t 37 371 31* * Serum insulin (pglml) 405.4 39.6 2056.4 43.0* 11,975.9 t 426.8* 6,175.0 t 355.8* 1,347.4 t 146.3 4,114.6 t 271.5* 11,615.8 ? 707.0* 2.020.8 t 69.8* * * + 'Group mean ( + SEM) values for control f /?I and diabetic mice that were either oil, estradiol(1 ~ 1 3 .days), 5 or progesterone (2 mgi3.5 days) treated starting a t 4 weeks of age. All measurements were made a t 8 and 16 weeks of age. Body weight, blood glucose, and serum insulin levels for all control mice, regardless of treatment, are represented as pooled values since no significant treatment-related differences were found. *Significant (PS 0.05) intergroup differences within age groups as compared to control values. expressed diabetes-obesity syndrome in C57BC/KsJ (dbldb) mice (Coleman et al., 1982, 1984; Garris, 1987a,b; Garris and Michel, 1988). Following chronic oral or injection therapies, such compounds as estradiol, dehydroepiandrosterone, and the opiate antagonist Nalmefene effectively normalize blood glucose levels or cellular glucose homeostasis to control values in (dbldb) mice (Coleman et al., 1982, 1984; Garris, 1987a,b; Garris and Michel, 1988). In addition estradiol therapy results in a resensitization of medial basal hypothalamic neurons to the modulating actions of progesterone (Garris and Coleman, 1984; Garris et al., 1985a-c), while Nalmefene normalizes regional brain glucose uptake rates (Garris and Michel, 1988). These studies, in addition to the recognized lipolytic actions of estrogenic or pre-estrogen steroids (Beck, 1973; Garris et al., 1986),suggest that the genomic expression of the diabetes-obesity syndrome in C57BL/KsJ mice may be suppressed, delayed, prevented, or modified by specific steroidal, or steroid-modified, receptor mechanisms (Barlett and Garris, 1987) that influence intracellular glucose metabolism (Garris et al., 1985a-c). The present studies were undertaken to determine the effectiveness of estrogen and progestin therapies on body weight, blood glucose, serum insulin, and lipoprotein lipase activity, and associated structural changes in the reproductive tract and peripheral tissues of 816-week-old female C57BL/KsJ (dbidb) mice following the overt expression of the type I1 diabetes-obesity syndrome. S.C.injections. The oil vehicle (0.1 ml) served a s the sham (control) injection procedure. Tissue Collection and Analysis The uteri were collected from 16-week-old diabetic mice following steroid treatment for analysis by transmission electron microscopy (TEM). As previously described (Garris, 1985; Garris et al., 1985a-q Garris et al., 19861, mice were anesthetized with sodium pentobarbital and perfused with 50 ml of physiological saline (by intracardiac puncture), which was followed by 100 ml of Karnovsky's fixative solution. The tissues were cleaned, blotted, blocked, and embedded in plastic by conventional techniques. All tissues were subsequently sectioned and stained with osmium tetroxide prior to examination by TEM. All uterine specimens examined were collected from the midcornua region. Serum Insulin Assay Serum insulin concentrations were assayed from duplicate samples by radioimmunoassay as previously described (Garris, 1988a) using mouse insulin (Novo Industries, Denmark: 17.2 IU/mg) standards. Assay sensitivity approximated 80 pg with a n intraassay variability of <lo%. All values were expressed a s pg/ ml. Tissue Lipoprotein Lipase Assay Following hormone replacement therapy, groups of 8- and 16-week-old mice were sacrificed by cervical disMATERIALS AND METHODS location and the ovaries and internal tissues removed Animals and weighed. Each tissue was immediately rinsed in Adult, female C57BL/KsJ mice (Jackson Laboratory, sterile Dulbecco's modified Eagle's (DME) medium conBar Harbor, ME) were used in these studies. Each nor- taining 2%bovine serum albumin, then blotted on stermal (+/?) mouse was age-matched with a genetically ile filter paper. The tissues were subsequently minced diabetic (dbldb) littermate. All mice were housed five and placed into sterile polypropylene culture tubes conper cage under controlled environmental (23°C) condi- taining 1 ml of DME medium supplemented with 2% tions, with a n established photoperiod of 12 h r light/ bovine serum albumin and 10 U of heparin. Tubes were day (lights on 0700 hr). Blood glucose levels (Ames then capped and incubated at 37°C in a 90% 02110% Glucometer method) and body weights were deter- CO2 atmosphere for 2 hr with gentle shaking. At the mined biweekly for all mice. Animals exhibiting both end of the incubation period, each tube was centrifuged obesity and hyperglycemia relative to controls (Table (3,OOOg/30min), and the supernatant was collected and used as the source of the tissue lipoprotein lipase in the 1)were regarded as overt diabetics. assays. Hormone Treatment Assays for lipoprotein lipase were performed as pre17-P-estradiol (E: 1pg/3.5 days) and progesterone (P: viously described (Garris et al., 1986), in triplicate, 2 mg/3.5 days) were dissolved in sesame oil (0.1 ml) for within 30 min after the preparation of the tissue lipase 312 D.R. GARRIS and P-treated controls relative to corresponding 8week-old mice (P<0.05), which was not attributable to a particular treatment regime. All control values were considered a s age-dependent and treatment independent, and are presented in such a manner for clarity (Table 1). At 8 weeks of age, (dbldb) mice exhibited a significant (Ps0.05) increase in body weight, blood glucose, and serum insulin levels in all treatment groups relative to age- and treatment-matched controls (Table 1). Although both E and P treatments moderated the expressed hyperglycemia in the (dbldb) group relative to oil-treated diabetics, the blood glucose levels remained elevated relative to corresponding controls. Serum insulin levels were significantly elevated in E- and Ptreated (dbldb) mice a s compared with oil-treated diabetics and all control groups (Table l). At 16 weeks of age (Table l),oil- and P-treated (dbl db) mice continued to exhibit obesity, hyperglycemia, and hyperinsulinemia relative to age- and treatmentmatched controls. However, E-treated (dbldb) mice exhibited normoglycemia as compared with controls, but continued to demonstrate obesity and marked hyperinsulinemia. The effects of E-therapy on serum insulin levels in (dbldb) mice was dramatic, resulting in a 2.8fold increase in oil-treated diabetic levels and elevated serum insulin levels 8.6-fold over those of corresponding controls. In contrast, P-treatment suppressed the age-related rise in serum insulin concentrations in 16 Statistical Analysis week old (dbldb) mice by approximately 50% a s comAll values were expressed as group means (2S.E.M.). pared with oil-treated diabetics. Intergroup differences with respect to genotype, age, Effects of Estradiol and Progesterone Therapy on Uterine and hormone treatment were analyzed using the StuEpithelial Structure in 16-Week-Old Diabetic Mice dent’s t-test, Newman-Keuls, or analysis of variance The effects of E and P modulation of systemic carboexams, where appropriate, with a P ~ 0 . 0 5accepted as representing statistical significance. hydrate regulatory parameters were correlated with structural changes in the uterine epithelium of (dbldb) Experimental Protocol mice induced by E- and P-treatments (Figs. 1, 2). The Starting a t 4 weeks of age, match-paired control ( + I steroid-sensitive epithelium of (dbldb) mice concen2) and (dbldb) C57BLIKsJ mice were treated with ei- trated fat in the form of lipid vacuoles, which were ther oil, E, or P every 3.5 days. Body weight and blood dispersed throughout the cytoplasm, with the highest glucose concentrations were monitored on a biweekly deposits localized a t the basal pole of the cells (Figs. basis. At either 8 or 16 weeks of age, mice were sacri- lA, 2A). A blunted, microvillus apical surface was obficed and tissues collected, a s indicated, for either served, as well as a n expanded basal membrane surface structural or functional indices and compared with extending into the underlying basal lamina layer, in body weight, blood glucose, and serum insulin concen- all (dbldb) specimens. Cytoplasmic organelles were trations. The effects of E and P treatments on uterine both sparse and indistinct, with a sparsity of mitochonstructure and associated reproductive tissue lipopro- dria and endoplasmic reticular organelles contrasting tein lipase activity were analyzed and compared with with a n expanded lipid vacuolar area. The nuclei of corresponding control parameters. these cells appeared to retain typical structural integrity. RESULTS Following P-treatment (Fig. lB), several of the diaEffects of Estradiol and Progesterone on Body Weight, betes-associated structural characteristics of the uterBlood Glucose, and Serum Insulin Levels in 8- and ine epithelium were modified. Lipid droplets failed to 16-Week-Old C57BUKsJ Mice stain as intensely and appeared to occupy a diminished The estrogen- and progestin-induced changes in con- cytoplasmic volume. Mitochondria, endoplasmic retictrol (+/?) and diabetic (dbldb) body weight, blood glu- ulum, and Golgi structures were readily identifiable in cose, and serum insulin parameters are summarized in P-treated epithelial specimens in contrast with oilTable 1. The 8- and 16-week-old control mice exhibited treated (dbldb) specimens. However, the apical surface similar intergroup values for each measured parame- continued to demonstrate blunted microvillus formater, with only age-related changes being noted. Control tion, whereas the basal membrane surface appeared body weights and blood glucose levels were comparable smooth. The nuclei of P-treated (dbldb) mice appeared between all 8- and 16-week-old treatment groups structurally similar to the corresponding oil-treated (P>0.05). In contrast, a consistent elevation in serum group. insulin concentrations was noted in all 16-week-old, EEstradiol therapy (Fig. 2B) induced a dramatic res- samples according to the method of Nilsson-Ehle and Schotz (Nilsson-Ehle and Schotz, 1976). In brief, 75 p1 of the isolated enzyme preparation was mixed with 25 pl of a substrate that consisted of 22.6 mM (3H)-triolein (1.4 pcilumol), lecithin (2.5 mglml), bovine serum albumin (40 mglml), 33% (vollvol) human serum, and 33% (vollvol) glycerol in 0.27 M Tris-HC1 (pH:8.1) buffer and incubated a t 37°C for 90 min. The reactions were subsequently terminated and the free fatty acids (FFA) were separated from the incubation mixture by liquid-liquid partition extraction as follows. To each tube, 3.25 ml of a methano1:chloroform:heptane (1.41: 1 . 2 5 1 vollvol) mixture was added, followed by 1.05 ml of a 0.1 M potassium carbonate-borate buffer (pH 10.5). The combined solution was vigorously mixed and centrifuged at 3,OOOg for 15 min at 23°C. An aliquot (300 pl) was obtained from the methanol-water upper phase, which contained the free fatty acids. Radioactivity present in the free fatty acid fraction was determined by liquid scintillation analysis. One milliunit of enzyme activity was defined as the release of 1nmol of fatty acid per 1min, and all values were then expressed as nanomoles FFAlminlgm tissue. The activity in all cases was inhibited by either the addition of 1M NaCl or by the omission of serum from the assay. All values were expressed as group means (kSEM) with intergroup differences (P <0.05) determined by the Student’s t-test for each tissue group analyzed. ESTRADIOL, PROGESTERONE, AND DIABETES 313 Fig. 1. Photomicrographs of uterine epithelial cells from 16-weekold, oil-treated (A: x 3,700) and progesterone-treated (B: x 4,800) diabetic (dbidb) mice. Both groups exhibited lipid (L) accumulation at the basal pole of the epithelial cell layer, with scattered lipid droplets being apparent in apical cytoplasmic zones above the nuclei (N). The basal lamina (BL) of the oil-treated, diabetic mice (A) exhibited pseudopodia-like evaginations into the the basement membrane, whereas the endometrial surface of progesterone-treated epithelial cells appeared smooth (B). There was no evidence of lipid accumulation in the periluminal stromal fibroblasts (S) in either treatment group. toration of structural organization to the uterine epithelial cells of (dbldb) mice. Lipid droplets were essentially absent from the epithelia of E-treated (dbldb) mice. In addition, restoration of organelle structure and organization, apical microvilli, and a smooth-contoured basal membrane surface were apparent. Nuclei remained structurally intact with a distinct nucleolus apparent in most. Thus, E-treatment was extremely effective in countering the diabetes-associated uterine epithelial adiposity t h a t characterizes reproductive tract dysfunction in the mutant murine model. vated LPL activity in (dbldb) mice as compared to oiltreated diabetics. The P-treated (dbldb) exhibited a depressed LPL activity as compared with oil-treated mice, demonstrating values which were comparable to P-treated controls. The ovarian samples at 8 weeks of age (Table 2) indicated that only P-treated control mice had detectable LPL values. In contrast, all (dbldb) groups exhibited detectable ovarian LPL activity, with E-therapy increasing, and P-treatment depressing, the diabetes-associated elevations in LPL relative to controls. At 16 weeks of age (Table 31, the LPL activity in both oil-treated control and diabetic groups had increased dramatically (P50.001) relative to corresponding 8week-old mice (Table 2). In addition, E-treatment induced a further elevation in LPL in both uterine and ovarian samples as compared with oil-treated controls. Chronic P-treatment suppressed the age-related rise in LPL activity in both ovarian and uterine tissues collected from control groups. In diabetic mice, both E- and P-treatments significantly limited the diabetes-associated increase in uterine LPL activity relative to oil-treated (dbldb) mice. However, only P-treatment effectively (P50.05) suppressed both ovarian and uterine LPL activity in (dbl db) mice to corresponding control values. These data Modulation of Lipoprotein Lipase by Estradiol and Progesterone in the Uterus and Ovaries of C57BUKsJ Mice The effects of E- and P-treatments on the diabetesassociated increase in uterine and ovarian lipoprotein lipase (LPL), a n enzyme that permits the uptake and storage of free fatty acids in non-hepatic tissues, are summarized in Tables 2 and 3. At 8 weeks of age (Table 2), uterine LPL was found to range between 1.2 and 2.0 nmol FFAlminlgm, regardless of hormone treatment, in all controls. In contrast, a significant diabetes-associated increase in LPL activity was observed in both oil- and E-therapy (dbldb) mice as compared to corresponding control groups. In addition, E-treatment ele- 314 D.R. GARRIS TABLE 2. Effects of estradiol and progesterone on tissue lipoprotein lipase activity in normal and diabetic C57BL/KsJ mice' Lipoprotein lipase Age Genotype N Treatment Tissue (n mol FFAlminigm) 8 ( + i?) 5 Oil Uterus 1.2 t 0.01 Uterus 2.0 2 0.5 5 E 8 Uterus 1.3 2 0.1 5 P 8 Uterus 7.1 * 0.9* 8 (dbldb) 5 Oil Uterus 11.8 t E 4 8 0.9 t 0.3' 4 P Uterus 8 Ovary ND ( + I?) 5 Oil 8 Ovary ND 8 5 E 8 5 P Ovary 0.2 t 0.02* 2.6 2 0.3* Ovary 8 (dbldb) 5 Oil Ovary 7.4 2 0.04%9* 8 4 E Ovary 1.3 2 0.032,* 4 P 8 O.l23* 'All values are expressed as group means ( +- SEM) for the indicated 8-week-old control ( + I?) and diabetic (dbldb) mice tissues following either oil, estradiol (E: 1 kg) or progesterone (P2 mg) treatment twice a week (i.e., every 3.5 days) starting at 4 weeks of age. All assays were run in triplicate from pooled samples for the indicated No. (N) of animals. Non-detectable, ND. 'Treatment-related differences within each genotype relative to oiltreated control values. *Genotype-related differences between similar treatment groups (Ps0.05). TABLE 3. Effects of estradiol and progesterone on tissue lipoprotein lipase activity in normal and diabetic C57BL/KsJ mice' Age Genotype N Treatment 16 ( + l?) 4 Oil 16 6 E 3 P 16 16 (dbldb) 5 Oil E 16 6 16 3 P 16 ( + l?) 4 Oil 16 6 E 16 3 P 16 (dbldb) 5 Oil E 16 6 16 3 P Lipoprotein lipase Tissue (nmol FFAlminlgm) Uterus 40.4 ? 2.8 Uterus 68.1 2 10.8% Uterus ND* Uterus 201.0 t 2.8* Uterus 105.0 t 12.B2.* Uterus ND* Ovary ND Ovary 342.0 t 49.0' Ovary ND Ovary 395.0 t 46.0* Ovary 431.0 t 72.0 Ovary ND' 'All values are represented as group means ( ? SEMI for the indicated control ( + /?I and diabetic (dbidb) mice tissues following oil, estradiol (E: 1 kg) or progesterone (P: 2 mg) treatment twice a week (i.e., every 3.5 days) starting at 4 weeks of age. All assays were run in triplicate from pooled samples from the indicated No. (N) of animals. Non-detectable, ND. 'Treatment-related differences in tissues from the same genotype groups relative to oil-treated values. *Genotype-relate differences (PsO.05)between groups receiving the same treatments. Fig. 2. Uterine epithelial cells from 16-week-old diabetic mice treated with oil ( A x 10,500) or estradiol ( B x 7,500). The obvious lipid (L) deposits present below the nuclear (N) zone in cells from oil-treated diabetic mice were absent in the estradiol-treated mice. The irregular contour of the basal lamina (BL) in the oil-treated, diabetic mice contrasted with the smooth basalar membrane surface of cells from estradiol-treated mice. The decline in the amount of intracellular lipid in estradiol-treated epithelial cells corresponded with the normalization of blood glucose levels in these groups relative to oil- and progesterone-treated diabetic mice. indicate a functional role for E and P in modulating intracellular lipid deposition in both uterine and ovarian tissues. DISCUSSION The results of the present studies indicate that ovarian steroid hormones and related pre-hormone steroids (Coleman et al., 1982, 1984; Garris, 1987a) effectively moderate the physical and cellular p a r a m e t e r s altered b y the o v e r t expression of the diabetes-obesity syn- ESTRADIOL, PROGESTERONE, AND DIABETES drome in C57BLlKsJ mice. The effectiveness of the steroidal therapies was directly related to their ability to modulate intracellular glucose and lipid homeostasis. The estrogen-induced normalization of utero-ovarian and peripheral tissue structure in (dbldb) mice, correlates with the correction of intracellular lipid metabolism. The normalization of these structural and functional indices suggests that the modulation of cellular compartments associated with lipid deposition and glucose utilization induces the tissue changes associated with the overt expression of the diabetes component of the diabetes-obesity syndrome in these species. Although E-therapy was effective in suppressing the hyperglycemia in (dbldb) mice, it was ineffective in suppressing either the obesity or hyperinsulinemia components of the syndrome. In contrast, P-therapy had no influence on blood glucose or body weight parameters, but did modify the hyperinsulinemia in 16-weekold (dbldb) mice relative to controls and oil-treated mutant mice. The specific actions of E- and P-treatments were demonstrated by the singular ability of E to restore normal intracellular structure to the uterine epithelial cells in (dbldb) mice, as donated by the prominence of intracellular organelles, surface modifications, and the apparent absence of intracellular lipid deposition (Garris, 1985; Garris et al., 1986). The restoration of utero-ovarian lipoprotein lipase activity to control levels following steroid therapy suggests that intracellular glucose metabolism, and not obesity or insulin receptor insensitivity (Barlett and Garris, 1988), is the primary functional cause for the diabetesassociated cellular atrophy in this species (Garris, 1985; Garris et al., 1985a-c, 1986). Since clinical studies demonstrate that similar structural and functional defects exist in female diabetics (Hall and Tillman, 1951; Krause, 1936; Williams and Porte, 1974), and that steroid-containing oral contraceptives markedly affect diabetes management (Bailey and Ahmed-Sorour, 1982; Gossain et al., 1983; Kalkhoff, 1972, 1982; Kalkhoff et al., 1970; Notelovitz, 1982; Paik et al., 19821,the results of the present studies may aid in explaining the mechanisms of action of steroids in moderating the expression of the diabetic condition in humans. Although both E and P treatments were demonstrated to reduced uterine lipoprotein lipase activity in uterine samples, only P reduced LPL activity in (dbldb) mice to control levels. In comparison, E proved to stimulate ovarian LPL in both control and diabetic groups at 16 weeks of age. These tissue-specific effects of E and P on this common parameter within the same female reproductive tract may reflect normal intrinsic differences in cellular responses by uterine and ovarian tissues to each hormone. While E is recognized to be actively mitogenic in uterine epithelial cells (Garris, 1984b; Kirkland et al., 19811, it serves as a negativefeedback suppressor of gonadatropin-stimulated follicular recruitment in the ovary (Barraclough, 1971; Kirchick et al., 1978). By comparison, P supports endometrial growth of E-primed endometrial cells (Garris, 1984b). However, without previous estrogenic stimulation, P suppresses uterine cellular growth and metabolism (Garris, 1984b). As such, the effectiveness of either steroid therapy may depend on the ability of a particular cell type to recognize and respond to the specific steroid. 315 The ability of ovarian steroid hormones to modulate intracellular lipoprotein lipase activity and the diabetes-associated elevation in LPL activity, supports and extends the results of previous studies with both type I (i.e., insulin-dependent) and type I1 (non-insulin-dependent) animal models (Garris et al., 1986; Gray and Greenwood, 1983, 1984). Estradiol treatment is recognized to alter cell lipase activity in the reproductive tract of rats (Gray and Greenwood, 1983, 1984), thus influencing the rate of triglyceride uptake by the uterus and ovary. As such, the depressed circulating E and P levels in the (dbldb) mouse (Garris, 1985; Garris et al., 1985a-c) may promote the diabetes-induced elevation in utero-ovarian LPL in this model (Garris et al., 1986). The therapeutic effectiveness of both steroidal agents in combating the diabetes-associated cellular changes thus appears to be dependent on 1)normal tissue or cellular sensitivity and responsivity to the individual hormones, 2) the effectiveness of each steroid in correcting intracellular carbohydrate utilization, and 3) the ability of E andlor P to modulate intracellular lipid catabolism and anabolism. Although the present studies focused on the intracellular structural and functional parameters modified by the diabetes-obesity syndrome in (dbldb) mice, similar cellular changes may relate to alterations in pancreatic, renal, hepatic and CNS tissue function in this model (Barlett and Garris, 1987; Bower et al., 1980; Bray and York, 1979; Coleman, 1967,1978,1982; Garris, 1984a, 1987a,b; Garris et al., 1984a,b, 1985a-c; Garris and Coleman, 1984; Garris and Michel, 1988; Gartner, 1979; Hanker et al., 1980; Herberg and Coleman, 1977; Johnson and Sidman, 1979; Like et al., 1974; Meade et al., 1981; Moore et al., 1980; Sima and Robertson, 1979). Regardless of the particular tissue, the longer the diabetic condition persists, the more pronounced and dramatic are the cellular alterations in this animal model. Previous studies showed that the genetic mutation does not account for the altered cellular activity or responsivity in (dbldb) mice (Garris, 1987b), but that the changes in cellular structure and function are a direct result of the hyperglycemic condition (Barlett and Garris, 1987; Garris, 1985, 1987a,b; Garris et al., 1985a-c). Recent studies (Barlett and Garris, 1988) have also indicated that insulin receptor binding in peripheral and central nervous system tissues is essentially the same in both control and diabetic mice. These data suggest that the hyperglycemic (but not the hyperinsulinemic) component of the diabetesobesity syndrome is the primary factor involved in the induction of peripheral tissue adiposity and atrophy in this animal model. The elevated glucose is hepatically modified and circulated as triglycerides (Garris et al., 1986) which are sequestered by peripheral tissues exhibiting elevated lipoprotein lipase activities. The elevation in intracellular lipid pools and content induce an imbalance in glucose homeostasis, resulting in the futile interaction of counterregulatory mechanisms as indicated by hyperadrenalcorticoid secretion (Coleman, 1967, 1978) and tissue noradrenergic activity (Garris, 198813). Ovarian steroids. through their selective modulation of cellular metak'olism in reproductive, pancreatic, hepatic, and CNS tissues, are capable of restoring (or inducing) normal ct?llular glucose metabolism (Garris, 1987a; Garris and Coleman, 1984; Gar- 316 D.R. GARRIS ris and Michel, 1988; Garris et al., 1984a,b, 1985a-c). As a result, lipid (triglyceride) production and deposition are reduced and structural parameters normalized in accordance with the specific therapeutic effects of the steroids. The demonstrated ability of ovarian steroid hormones, and related pre-steroid hormones, to influence similar cellular and tissue parameters in a variety of diabetic and obese animal models and clinical situations (Garris, 1988a; Garris et al., 1986; Gray and Greenwood, 1983) suggests that these agents have an enormous therapeutic potential. Previous studies have demonstrated that age-related changes in glucose uptake rates characterize the severity of the diabetesobesity syndrome in (dbldb) mutant mice (Garris, 1985; Garris and Michel, 1988; Garris et al., 1984a,b, 1985a-c). During the early phase of the genomic expression, most peripheral tissues demonstrate an enhanced glucose uptake under hyperglycemic and hyperinsulinemic states (Garris et al., 1985a-c). With age, and the onset of progressive diabetes-related cellular alterations in structure and function (Coleman, 1967, 1978; Garris, 1985; Garris et al., 1985a-c, 1986), glucose uptake rates decline in association with tissue atrophy, adiposity, or premature degeneration (Garris et al., 1986). In the present studies, the initiation of the E- and P-treatment regimes coincided with the expected onset of the obesity, hyperinsulinemia, and hyperglycemia components of the syndrome at 4 weeks of age in this animal model (Coleman, 1967, 1978). The results suggest that the longer the therapeutic regimes were continued (i.e., to 16 weeks of age vs. 8 weeks of age), the more effective the treatments were in suppressing the changes noted in the structure and metabolic parameters of oil-treated (dbldb) mice. It remains to be determind if the initiation of such therapeutic regimes prior to the overt expression of the genomic mutation in this animal model would be effective in suppressing the diabetes-related cellular and tissue manifestations. In summary, the diabetes-associated alteration in utero-ovarian structure and function in C57BLlKsJ (dbldb) mice were responsive to the therapeutic actions of E and P. The ability to normalize selectively both structural and intracellular metabolic parameters related to glucose and lipid homeostasis, suggests that the diabetes-associated cellular changes characterizing this and related diabeticlobese animal models (Garris, 1984a, 1988a,b; Garris et al., 1984a,b; Gray and Greenwood 1983,1984) may be either corrected, delayed, prevented, or suppressed through the use of steroidal, therapeutic agents. ACKNOWLEDGMENTS The author expresses his sincere appreciation to S.K. Williams, W. Bowman, D.S. Whitehead, and C. Cook for their excellent technical assistance during various phases of these studies. The support and advice of Drs. D.L. Coleman and P. H. Pekala during the course of these studies are gratefully acknowledged. LITERATURE CITED Bailey, C.J., and H. Ahmed-Sorour 1982 Role of ovarian hormones in the long-term control of glucose homeostasis: Effects on insulin secretion. Diabetoloaia. 19:475-481. Barlett, P.B., and D.R. Girris 1987 Diabetes-associated alterations in cytosolic estradiol receptor binding on the peripheral tissues of C57BLiKsJ mice. Med. Sci. Res., 15:1421,1422. Barlett, P.B., and D.R. Garris 1988 The diabetes-obesity syndrome does not influence insulin receptor binding on uptake rates in the brain or peripheral tissues of C57BL/KsJ (dbldb) mice. Med. Sci. Res., 16:133,134. Barraclough, C.A. 1971 Sex steroid regulation of reproductive neuroendocrine processes. In: R.O. Greep, ed. Handbook of Physiology, Physiological Society, Washington, D.C.vol. 2, sect. 7. pp. 29-56. Beck, P. 1973 Contraceptive steroids: Modifications of carbohydrate and lipid metabolism. Metabolism, 22t841-855. Bower, C . , B.M. Brown, M.W. Steffes, R.L. Vernier, and S.N. Mauer 1980 Studies of the glomerular mesangium and the juxtaglomerular apparatus in the genetically diabetic mouse. Lab. Invest., 43t333-346. Bray, G.A., and D.A. York 1979 Hypothalamic and genetic obesity in experimental animals: An autonomic and endocrine hypothesis. Physiol. Rev., 59:719-809. Coleman, D.L. 1967 Studies with the mutations diabetes in the mouse. Diahetologia, 3:238-248. Coleman, D.L. 1978 Obesity and diabetes: Two mutant genes causing diabetes-obesity syndromes in mice. Diahetologia, 14:141-148. Coleman, D.L. 1982 Diabetes-obesity syndromes in mice. Diabetes, 3 I ( S ~ p p l1):1-6. . Coleman, D.L., E.H. Leiter, and R.W. Schwizer 1982 Therapeutic effects of dehydroepiandrosterone (DHEA) in diabetic mice. Diabetes, 31:830-833. Coleman, D.L., E.H. Leiter, and R.W. Schwizer 1984 Effects of genetic background on the therapeutic effects of dehydroepiandrosterone (DHEA) in diabetes-obesity mutants and in aged normal mice. Diabetes, 33:26-32. Garris, D.R. 1984a Effects of progressive hyperglycemia on ovarian structure and function in the spontaneously diabetic Chinese hamster. Anat. Rec., 21Ot485-489. Garris, D.R. 198413 Postpartum regeneration of the guinea pig endometrium: Relationship to serum estradiol and progesterone concentrations. Anat. Rec., 210:41-44. Garris, D.R. 1985 Diabetes-associated alterations in uterine structure in the C57BLIKsJ mouse: Relationship to changes in estradiol accumulation, circulating ovarian steroid levels and age. Anat. Rec., 211:414-419. Garris, D.R. 1987a Depressed progesterone accumulation by the brain and peripheral tissue of diabetic C57BLIKsJ mice: Normalization by estrogen therapy. Horm. Res., 25:37-48. Garris, D.R. 19871, Obese (obiob) and diabetes (db/dh) mutations: Two factors modulating brain and peripheral tissue uptake of estradiol in C57BLIKsJ mice. Dev. Brain Res., 35t153-157. Garris, D.R. 1988a Effects of diabetes on uterine condition, decidualization, vascularization and corpus luteum function in the pseudopregnant rat. Endocrinology, 122:665-672. Garris, D.R. 198813Reproductive tract and pancreatic norepinephrine levels in pre- and overt-diabetic C57BLiKsJ mice: Relationship to body weight, blood glucose, serum insulin and reproductive dysfunction. Proc. Soc. Exp. Biol. Med., 189r79-83. Garris, D.R., and D.L. Coleman 1984 Diabetes-associated changes in estradiol accumulation in the aging C57BL/KsJ mouse brain. Neurosci. Lett., 49:285-290. Garris, D.R., D.L. Coleman, and C.R. Morgan 1985 Age- and diabetesrelated changes in glucose uptake and estradiol accumulation in the C57BLiKsJ mouse. Diabetes, 34:47-52. Garris, D.R., and M.E. Michel 1988 Regional glucose uptake in genetically diabetic C57BLIKsJ mice: Modulation by the opiate antagonist, Nalmefene. Brain Res., 445:262-267. Garris, D.R., R.L. West, and D.L. Coleman 1985 Morphometric analysis of medial basal hypothalamic neuronal degeneration in diabetes (dbidh) mutant C57BL/KsJ mice: Relation to age and hyperglycemia. Dev. Brain Res., 2Ot161-168. Garris, D.R., R.L. West, and P.H. Pekala 1986 Ultrastructural and metabolic changes associated with reproductive tract atrophy and adiposity in diabetic female mice. Anat. Rec., 216t359-366. Garris, D.R., S.K. Williams. and D.L. Coleman 1984 Glucose utilization in the mouse brain: Influence of age and diabetes. Dev. Brain Res.. 15t141-146. Garris, D.R., S.Williams, C. Smith-West, L. West 1984 Diabetesassociated endometrial disruption in the Chinese hamster: Structural changes in relation to progressive hyperglycemia. Gynecol. Obstet. Invest., I7t293-300. Garris, D.R., S.K. Williams, and L. West 1985 Morphometric evaluation of diabetes-associated, ovarian atrophy in the C57BLIKsJ mouse: Relationship to age and ovarian function. Anat. Rec., 21: 434-443 ESTRADIOL, PROGESTERONE, AND DIABETES Gartner, K. 1979 Glomerular hyperfiltration during the onset of diabetes in mice. Diabetologia, 15t59-63. Gossain, V.V., N.K. Sherma, A.M. Mickelakis, and D.R. Rovner 1983 Effect of oral contraceptives on plasma glucose, insulin and glucogen levels. Am. J . Obstet. Gynecol., 147:618-623. Gray, J.M., and M.R.C. Greenwood 1983 Uterine and adipose lipoprotein lipase activity in hormone-treated and pregnant rats. Am. J. Physiol., 245:E132-E137. Gray, J.M., and M.R.C. Greenwood 1984 Effect of estrogen on lipoprotein lipase activity and cytoplasmic progestin binding sites in lean and obese Zucker rats. Proc. Soc. Exp. Biol. Med., 175:374379. Hall, R.E., and A.J.B. Tillman 1951 Diabetes and pregnancy. Am. J. Obstet. Gynecol., 61:1107-1115. Hanker, J.S., W.W. Ambrose, P.E. Yates, G.G. Koch, andK.A. Carson 1980 Peripheral neuropathy in mouse diabetes mellitus. Acta Neuropathol. (Berl.), 51t145-154. Herberg, L., and D.L. Coleman 1977 Laboratory animals exhibiting obesity and diabetes syndromes. Metabolism, 26.59-98. Johnson, L.M., and R.L. Sidman 1979 A reproductive endocrine profile in the diabetes (db) mutant mouse. Biol. Reprod., 20t552559. Kalkhoff, R.K. 1972 Effects of oral contraceptive agents and sex steroids on carbohydrate metabolism. Annu. Rev. Med., 23:429438. Kalkhoff, R.K. 1982 Metabolic effects of progesterone. Am. J . Obstet. Gynecol., 142.735-738. Kalkhoff, R.K., M. Jackson, and D. Lemper 1970 Progesterone, pregnancy and the augmental plasma insulin response. J. Clin. Endocrinol., 31:24-34. Kirchick, H.J., P.L. Keyes, and B.E. Frye 1978 Etiology of anovulation in the immature alloxan-diabetic rat treated with pregnant 317 mare’s serum gonadotropin: Absence of the preovulatory luteinizing hormone surge. Endocrinology, 109t316-318. Kirkland, J.L., G.N. Barrett, G.M. Stance1 1981 Decreased cell division of the uterine lumina1 epithelium of diabetic rats in response to 17-B-estradiol. Endocrinology, 109t316-318. Krause, J. 1936 Diabetes mellitus und Schwangerschaft. Med. Klinik., 12t375-378. Like, A.A., R. Lavine, L. Paffenbarger, and W. Chick 1974 Studies in the diabetic mutant mouse. VI. Evolution of glomerular lesions and associated proteinuria. Am. J . Pathol., 66.193-207. Meade, C.S., D.R. Brandon, W. Smith, R.G. Simmons, S. Harris, and C. Sowter 1981 The relationship between hyperglycemia and renal immune complex deposition in mice with inherited diabetes. Clin. Exp. Immunol., 43t109-120. Moore, S.A., R.G. Peterson, D.L. Fellow, T.R. Cartwright, B.L. OConnor 1980 Reduced sensory and motor conductance velocity in 24-wk-old diabetic (C57BLIKs dbidb) mice. Exp. Neurol., 70: 548-555. Nilsson-Ehle, P., and M.C. Schotz 1976 A stable radioactive substrate emulsion for assay of lipoprotein lipase. J . Lipid Res., 17.536541. Notelovitz, M. 1982 Carbohydrate metabolism in relation to hormonal replacement therapy. Acta Obstet. Gynecol. Scand. [Suppl.l, 106: 51-56. Paik, S.G., M.A. Michelis, Y.T. Kim, and S. Shin 1982 Induction of insulin-dependent diabetes by streptozotocin: Inhibition by estrogens and potentiation by androgens. Diabetes, 31:724-729. Sima, A., and D.M. Robertson 1979 Peripheral neuropathy in the mutant diabetes mouse BUKs dbidb. Acta Neuropathol. (Ber1.1, 41:85-89. Williams, R.H., D. Porte, J r . 1974 The Pancreas, In: Textbook of Endocrinology. R.H. Williams, ed. W.B. Saunders, Philadelphia, pp. 502-626.