db) mutation-associated utero-ovarian involution: counter-regulatory influences of progesterone

db) mutation-associated utero-ovarian involution: counter-regulatory influences of progesterone

Pathophysiology 11 (2004) 41–50 Hypercytolipidemia promotes diabetes (db/db) mutation-associated utero-ovarian involution: counter-regulatory influen...

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Pathophysiology 11 (2004) 41–50

Hypercytolipidemia promotes diabetes (db/db) mutation-associated utero-ovarian involution: counter-regulatory influences of progesterone David R. Garris∗ , Bryan L. Garris Division of Cell Biology and Biophysics, Schools of Biological Sciences and Medicine, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA Received 6 October 2003; received in revised form 20 January 2004; accepted 18 February 2004

Abstract Background: The diabetes (db/db) mutation induces a hyperglycemic–hyperinsulinemic endometabolic environment that promotes hypercytolipidemic, utero-ovarian involution in C57BL/KsJ mice, resulting in reproductive sterility and eventual organoatrophy. Objective: Evaluation of the effectiveness of progesterone therapy (P-HRx), initiated prior to the genetic expression of the overt diabetes–obesity syndrome (DOS), on moderating the severity of female reproductive tract involution promoted by db/db mutation expression was evaluated by analysis of cytoarchitectural, endocrine and tissue lipo-metabolic indices relative to oil (O)-vehicle-treated (HRx) control (+/?) and db/db groups. Experimental design: All HRx treatments were started at 4 weeks of age (pre-overt DOS stage) and continued through 16 weeks of age (chronic DOS expression) when tissue and cellular endometabolic parameters were evaluated. Results: The DOS induced a dramatic increase in phenotypic obesity, hyperglycemia and hyperinsulinemia in db/db groups, relative to +/?, throughout the experimental period. In contrast, utero-ovarian weights were dramatically reduced in db/db groups relative to +/? indices. Chronic P-HRx effectively reversed these DOS-induced trends in db/db groups, maintaining moderated body and tissue weights, as well as re-establishing normal insulin indices, under a persistent hyperglycemic condition. In addition, P-HRx moderated the dramatic hypercytolipidemic condition which promotes utero-ovarian involution in db/db mice as evidenced by the reduction in observed tissue cytolipidemia. The concurrent normalization of tissue lipase and enhancement of glucose utilization indices by db/db utero-ovarian compartments, under moderated insulin recognition parameters, indicated that P-HRx effectively suppressed the severity of both the structural and endometabolic consequences of the DOS in db/db groups, without restraining hyperglycemic conditions. Conclusion: These results indicate that the pathophysiological alterations induced by the db/db mutation may be modulated through low-dose steroidal therapy, the efficacy of which is suspected to occur by the augmentation of normal insulin-coupled, post-receptor directed glucose utilization via the stimulation of oxidative metabolic pathways capable of maintaining normal utero-ovarian structural continuity and metabolic homeostasis. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Diabetes-induced reproductive tract atrophy; Hypercytolipidemia; Utero-ovarian involution; Progesterone therapy; Lipotoxicity; Diabetes (db/db) mutation; Hyperinsulinemia; Uterine epithelial tissue; Ovarian folliculogenesis

1. Introduction Organoatrophy of the female reproductive tract is a consequence of the expressed diabetes–obesity syndrome (DOS) associated with the expression of the diabetes (db/db) mutation in C57BL/KsJ mice [1–4]. Characterized by a progressive hypercytolipidemia-induced transformation ∗ Corresponding author. Tel.: +1-816-235-5857; fax: +1-816-235-6563. E-mail address: [email protected] (D.R. Garris).

of utero-ovarian tissue compartments into adipocyte-like domains [2,4], a dramatic compromise of the structural and endophysiological aspects of reproductive function precede eventual tissue dysfunction and pronounced involution [2,3]. Suppressed ovarian folliculogenesis and steroidogenesis [4], enhanced endometrial adiposity and hypo-vascularization [2] as well as utero-ovarian compartmental metabolic shifts from normal oxidative to lipogenic non-oxidative dominance [2,5–7], promote female reproductive incompetence. The resulting sterility occurs under hyperglycemic–hyperinsulinemic, Type II (NIDDM) en-

0928-4680/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2004.02.001


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dometabolic conditions, expressed with overt phenotypic obesity and accompanying hypertriglyceridemia [1,2]. The combination of these multiple endocrine-related, pathophysiological events being expressed concurrently, as well as a mutation-based absence of cellular leptin receptor responsivity, results in the structural and functional demise of the female reproductive tract in (db/db) mutants prior to the expected onset of the normal pubertal expression of utero-ovarian growth and development, as demonstrated by normal (+/?) C57BL/KsJ mice [2,6]. Recent studies have indicated that the severe compromise of utero-ovarian development in (db/db) mutants may be prevented if gonadal steroid therapy or stimulation is initiated prior to the pubertal-onset of the DOS in C57BL/KsJ (db/db) females [8,9]. Low-dose, estradiol (E) therapy has been demonstrated to effectively suppress the (db/db)-induced utero-ovarian adiposity which promotes organoatrophy in this experimental model [8,9]. In addition, the utilization of either pre-steroidal or post-cholesterol metabolites has been demonstrated to ameliorate the severity of the expressed-DOS in C57BL/KsJ mice, but not as effectively as ovarian steroid replacement therapy (HRx) [8–12]. However, when administered either enterically or by injection, the hyperglycemic–hyperinsulinemic components of the DOS are moderated in the presence of persistent obesity [8,9,12]. Of particular interest has been the noted preservation of normal cytoarchitecture in the utero-ovarian tissue compartments of E-HRx females when treatments commenced prior to the expression of the DOS in (db/db) mutants [9]. Moderated obesity, normoglycemia, reduced cytolipidemia and the suppression of tissue lipogenic indices maintained reproductive cytoarchitecture comparable to that of (+/?) controls [2,8,9]. These data suggested that ovarian steroid HRx may prove an effective method of maintaining reproductive vitality, even under conditions of persistent hyperinsulinemia. To date, however, the efficacy of progesterone (P)-HRx on moderating the severity of the DOS [13] on utero-ovarian tissue indices recognized to be influenced by the Type II diabetic condition has not been reported in this genetically-mutant (db/db) model, even though adrenergic counter-regulatory responses to DOS conditions have been recognized to be modulated by P-HRx [6,9]. The present studies define the influence of P-HRx on moderating the influences of the DOS-induced endometabolic aberrations on the phenotypic, utero-ovarian cytoarchitectural, endocrine and metabolic indices recognized to adversely promote reproductive incompetence through expression of the db/db mutation in C57BL/KsJ mice.

2. Materials and methods 2.1. Animals Adult, female C57BL/KsJ mice (Jackson Laboratory, Bar Harbor, ME) were used in these studies. Littermate con-

trol (+/?) and diabetic (db/db) genotypes were matched for phenotype, tissue sampling, blood glucose and serum insulin concentration comparisons, with each parameter monitored as an indicator of the severity of the expressed DOS. All mice were housed five per cage under controlled environmental conditions (23 ◦ C), with an established photoperiod of 12 h:12 h light/day (lights on: 06:00 h). Blood glucose levels (Ames Glucometer method) [14], the radioimmunoassay (RIA) analysis of serum insulin (Novo Industries, Denmark: 17.2 IU/mg standards) [7], analysis of tissue lipoprotein lipase activity [1,2], as well as tissue and body weight changes, were monitored in 4 (i.e., pre-overt DOS expression stage)- to 16 (i.e., chronic DOS expression stage)-week-old (db/db) and (+/?) groups. Animals exhibiting both obesity (≥20 g by 4 weeks of age; controls ≤ 17 g) and pronounced hyperglycemia (≥200 mg/dl) relative to controls (≤150 mg/dl) were considered as overt diabetics [11,12] while the progressive expression of these (db/db) mutation indices were monitored between 4 and 16 weeks of age [2]. 2.2. Progesterone treatments Progesterone (P: 1 mg/3.5 days; Sigma) was dissolved in sesame oil (0.1 ml) for subcutaneous injections. The oil vehicle (0.1 ml) served as the sham-control injection procedure as previously described [8,9]. 2.3. Serum insulin assay Serum insulin concentrations were evaluated from duplicate pooled serum samples by radioimmunoassay as previously described and validated [6] using mouse insulin (17.2 units/mg: Novo Industries) standards [7]. Assay sensitivity approximated 80 pg with an intra-assay variability of <10%. All serum insulin values were expressed as pg/ml. 2.4. Utero-ovarian

125 I-insulin

binding analysis

Tissue 125 I-insulin binding rate determinations were performed as previously described [8,15] utilizing monoiodinated insulin (porcine: 107 ␮Ci/␮g specific activity; New England Nuclear, Boston, MA) purified by ion exchange chromatography and diluted to an injection concentration of 1.5 ␮Ci/0.15 ml with physiological saline. In brief, a 10 ␮Ci iv-pulse of E was allowed to reach circulating equilibration for 30 min to obtain maximal tissue labeling as previously determined [15]. At 30 min post-injection, a 0.1 ml aliquot of blood was collected from each animal followed by subsequent intra-cardiac perfusion with 10cc of saline for removal of unbound insulin. Uterine and ovarian tissues were isolated, removed, cleaned and weighted to the nearest 0.1 mg then dissolved in 1 ml of Scintigest tissue solubilizer (Fisher) and 0.2 ml of water added to each sample vial [16,17]. Each sample was allowed to dissolve for 18 h at

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50 ◦ C, then subsequently added to 15 ml of scintillation fluid, mixed and 125 I-insulin uptake determined and expressed as utero-ovarian tissue 125 I-insulin binding/mg protein/30 min [15].


uration concentration, with affinity Kd values determined from displacement curves. All membrane receptor values were expressed as fmol/mg protein. 2.6. Tissue glucose uptake rate analysis

2.5. Tissue membrane insulin receptor binding analysis Insulin receptor-specific binding analysis was performed on utero-ovarian tissue membrane preparations collected from the specified treatment groups as previously described and validated [9]. In brief, each tissue sample was homogenized in a Tris-buffered sucrose solution, membrane preparations collected by centrifugation separation and re-suspended in homogenate buffer to 8 mg protein/ml. Insulin receptor binding was estimated using 10−6 M 125 I-insulin (porcine: 10 ␮Ci/␮g specific activity) incubated with 200 ␮g of membrane protein. The binding reaction was stimulated in a shaking water bath at 37 ◦ C for 20 min and terminated by placing the tubes on ice. Non-specific binding (20–25%) was determined by triplicate incubations in the presence of 10–5 radioinert insulin. Membrane-bound ligand was separated from bound by rapid filtration through Whatman (Clifton, NJ) GFC glass fiber filters, and each membrane preparation prepared rinsed twice with 5 ml of cold buffer. Filters containing the membrane-bound radioligand were dried and the radioactivity measured using a Beckman Gamma counter. Total insulin receptor numbers were determined from equilibrium binding curves at a sat-

Radiolabeled 1,2-3 H[N]2-deoxyglucose (specific activity: 37.3 Ci/mM; New England Nuclear) was purified by thin-layer chromatography and used to determined utero-ovarian glucose uptake rates as previously described [7,18]. From each tissue and blood sample, the tissue-specific glucose uptake rate was calculated, corrected for systemic blood glucose levels and specific activity, and expressed as ␮mol glucose utilized/mg protein/30 min. 2.7. Utero-ovarian tissue lipoprotein lipase In order to relate the recognized diabetes-induced changes in cellular and tissue structural indices to concomitant alterations in cellular metabolic status, uterine and ovarian tissue samples were collected from both oil- and P-HRx-treated, 16-week-old control (+/?) and diabetic (db/db) groups for analysis of tissue lipid-synthesis activity (triacylglycerol lipase). Tissue lipoprotein lipase activity was measured as previously described [1,2,8] from triplicate pools of uterine and ovarian samples collected from the 16-week-old (+/?) and (db/db) treatment groups. All group mean (±S.E.M.) values were expressed as nanomoles FFA/min/mg protein.

Fig. 1. Body weights (g) of control (+/?) and diabetic (db/db) C57BL/KsJ mice between 4 and 16 weeks of age are demonstrated as group means (N = 5–8 per group) and the influences of either oil-vehicle (0.1 ml/3.5 days sc) or P-HRx injections (2.0 mg/3.5 days sc) denoted. No significant differences existed between either (+/?) treatment group, receiving either oil or P injections, throughout the experimental period. All (db/db) mice, treated with oil or P-HRx, demonstrated body weights which were significantly (P ≤ 0.05) greater than comparable (+/?) treatment groups (∗). In addition, between weeks 8 and 16, all (db/db)-P-HRx mice demonstrated a significant decline in body weights relative to oil-HRx-(db/db) mice (#).


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Table 1 Progesterone modulation of diabetes-induced utero-ovarian hypercytolipidemia and endometabolic indices in C57BL/KsJ (db/db) mice Group Diabetes (db/db) mutation

Progesterone therapy

Index comparison

Relative to (+/?) controls

Relative to (db/db) mutants

Parameter Body weight Uterine weight Ovarian weight Tissue cytology

Pronounced obesity Atrophic Suppressed Hypercytolipidemic

Moderated (10–30%) Normalized Normal Moderate cytolipidemia

Endometabolic indices Blood glucose Serum insulin Tissue insulin binding indices Tissue insulin receptor population Lipid metabolism Carbohydrate metabolism

Hyperglycemia Hyperinsulinemia Increased Normal Enhanced Enhanced

Hyperglycemia Moderated Enhanced Normal Reduced Stimulated

Summation of the primary diabetes-associated indices moderated by progesterone therapy in C57BL/KsJ (db/db) mice relative to recognized control (+/?) parameters as described.

2.8. Tissue collection and preparation for cytochemical analysis Uteri and ovaries from 16-week-old control (+/?) and diabetic (db/db) matched-paired groups, treated with either oil-vehicle or P-HRx between 4 and 16 weeks, were collected and prepared for cytochemical analysis of tissue and cellular lipid deposition as previously described [2,4]. In brief, mice were anesthetized at the designated age-related experimental stages of DOS (db/db) mutation expression with sodium pentobarbital and systemically perfused with

50 ml of physiological saline and 100 ml of Karnovsky’s fixative solution. Uterine and ovarian tissue samples were cleaned, blotted, blocked and embedded in plastic using conventional techniques as previously described [4]. All tissue samples were subsequently sectioned and stained for polychromatic intra-cytoplasmic lipid identification [2,19] and localization using high-resolution light microscopic (HRLM) examination. Cytochemical analysis of the tissue sections prepared for light microscopic analysis were used for cytoplasmic polychromatic organelle designation, localization of intra-cellular lipid accumulations, and for

Fig. 2. Uterine and ovarian tissue weights at 16 weeks of age are depicted for control (+/?) and diabetic (db/db) C57BL/KsJ mice following either oilor P-HRx between 4 and 16 weeks of age. Both uterine and ovarian weights of (db/db)-oil-treated group were significantly (∗ P ≤ 0.05) lower than those of comparable (+/?) controls. The P-HRx induced a significant increase in uterine weights in (db/db) mice relative to oil-HRx (#) and restored (db/db) tissue weights to that of oil-HRx (+/?) groups. P-HRx did not stimulate (db/db) ovarian weight increases relative to those demonstrated by (+/?)-P-HRx groups. (∗) Significant intergroup differences (P<0.05) relative to genotype. (#) Significant intra-group differences relative to treatment regime.

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the subsequent determination of cytoplasmic lipid density alterations which characterize progressive DOS cytological indices, as previously described [1–4]. Photographic images of uterine endometrial epithelial tissue, as well as ovarian interstitial, thecal and follicular granulosa cell/tissue compartments, from the prepared tissue samples were captured with an Olympus (Olympus Optical, Tokyo, Japan) digital graphics camera and microscope unit. Subsequently, identified lipid vacuole pools were digitally enhanced utilizing polychromatic stain identification [19] and digital conversion for chemical-specific, scale imaging analysis of cytoplasmic lipid distribution and density determinations using a computer assisted photodigital scanner as previously described [2]. All cytoplasmic lipid pool changes associated with db/db-induced, progressive cytolipidemia were analyzed by HRLM and digital chemospectrophotographic (DCSP) analysis as described [2] in order to define inter- and intra-cellular lipid migration patterns and changes in progressive, DOS-induced, utero-ovarian tissue compartmental hypercytolipidemia [4]. Tissue sections prepared for DCSP image analysis of structural variations in cellular integrity, cytoplasmic changes in organelle and lipid inclusion density, as well as for trans-laminal epithelial and peri-follicular (i.e., thecal and interstitial) changes associated with the (db/db) mutation-induced imbibition of interstitial lipid deposits during the designated, age-related stages of the DOS were performed as previously described [2].


tissue weights in +/? groups, it promoted a normalization of uterine weights in the db/db mice (Fig. 2), but did not ameliorate the DOS-suppressive influence on ovarian mass (Table 1). The concurrent expression of systemic hyperglycemia (Fig. 3) in db/db groups relative to controls was not moderated by the P-HRx therapy. However, the db/db expressed hyperinsulinemia was dramatically reduced by 50% in 16-week-old groups, a systemic reduction comparable to that noted at the 4-week onset-phase of the DOS. Thus, the moderation of phenotypic obesity and the correction of utero-ovarian tissue mass to near +/? indices both occurred under moderated insulinemic, but persistent hyperglycemic, systemic endometabolic conditions in (db/db) following P-HRx (Table 1). Utero-ovarian cytoarchitecture was dramatically altered by the expression of the DOS condition in db/db mice relative to +/? groups. Uterine endometrial epithelial cells exhibited a marked hypercytolipidemia, relative to +/? specimens, at 16 weeks of age (Fig. 4A and B). Following digital

2.9. Statistical analysis Values for body weight, blood glucose and serum insulin levels, as well as tissue endometabolic indices, were expressed as group means (±S.E.M.) for the designated age, genotype and HRx groups for both control (+/?) and diabetic (db/db) mice. Intergroup differences with respect to genotype or HRx comparisons were determined using the Student’s t-test, Newman–Keuls or analysis of variance exams, where appropriate, with a P ≤ 0.05 accepted as representing statistical intergroup measurement differences.

3. Results The expression of the db/db mutation resulted in a dramatic and sustained phenotypic obesity between 4 and 16 weeks of age relative to (+/?) groups (Fig. 1). Body weights increased in a linear fashion in db/db mice between 4 and 14 weeks of age, approximating a three-fold increase in body mass relative to +/? mice. The biweekly P-HRx treatments had no demonstrable effect on body weights in +/? groups, but moderated a 10–30% decrease in body mass between 8 and 16 weeks of age in db/db groups (Fig. 1; Table 1). In contrast, utero-ovarian tissue weights were suppressed in db/db groups, relative to +/?, at 16 weeks (Fig. 2). In addition, although P-HRx did not influence

Fig. 3. Blood glucose (mg/dl) and serum insulin (pg/ml) concentrations are represented as group means from control (C) and (db/db) groups (N: 5–8 per group) at 16 weeks of age following either oil- or P-HRx treatments. (∗) Significant intergroup differences between (+/?) and (db/db) mice treated with either oil vehicle or P-HRx injections. The influence of P-HRx on intra-group differences for these parameters is denoted as (#).


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Fig. 4. Photomicrographs (1000×) of the uterine epithelial endometrial tissue layer, demonstrated by high-resolution light (A and B) or digital chemospectrophotometry for cytoplasmic lipid depositions (C–F), collected from 16-week-old control (+/?: A, C, E) and diabetic (db/db: B, D, F) mice following either oil (A–D) or P-HRx (E and F) treatments. The lipid deposition (arrows) noted in the basal pole of (+/?) uterine epithelial cells (A) was markedly enhanced by the DOS expressed in (db/db) mice (B). Examination by DCSP depicts the intra-cytoplasmic lipid pools as fluorescent yellow deposits at normal concentrations in controls (+/?: C) and elevated densities in diabetics (db/db: D), including apical cytoplasmic lipid depositions. Following P-HRx, the DCSP localization of lipid accumulations in both the (+/?: E) and (db/db: F) groups had been moderated to that observed in oil-HRx controls (C), indicating the cytolipid-suppressing effectiveness of the P-HRx therapy in diabetic groups.

cytolipid enhancement, the normal cytoplasmic lipid vacuole distribution and density patterns observed in normal tissue samples (Fig. 4C) was recognized to be remarkably expanded in age-comparable db/db tissue (Fig. 4D) samples. Following P-HRx (Fig. 4E and F), the normal basal pole lipidemia present in +/? samples remained unaffected, whereas the distribution and density of the cytolipid deposits were moderated in the db/db groups. Ovarian interstitial and thecal tissue compartments demonstrated enhanced cytolipidemia in all db/db groups relative to +/? indices (Fig. 5A and D). Following P-HRx, the density and intensity of cytolipid depositions was reduced in db/db mice, but remained present in +/? groups. These data indicated that the moderated hyperinsulinemia in db/db groups by the P-HRx treatments was associated with the reduced severity of the utero-ovarian hypercytolipidemia that typified reproductive organoinvolution under DOS conditions (Table 1). Alterations in tissue metabolic indices related to lipogenesis and insulin-dependent glucose utilization were modified by P-HRx therapy. The db/db mutation promoted a rapid and sustained elevation in tissue lipoprotein lipase activity in both uterine and ovarian tissues relative to +/? samples (Fig. 6). Following sustained P-HRx, utero-ovarian lipase activities were significantly suppressed in utero-ovarian samples from db/db groups relative to +/? indices (Fig. 6). The suppressive actions of P on tissue lipogenic activity

was also noted to occur in +/? tissue samples, indicative of the universal actions of the steroid on lipogenesis. The suppression of the non-oxidative lipid metabolic parameters was accompanied by a stable tissue membrane insulin receptor population and binding in all groups (Fig. 7A), but enhanced total insulin binding in db/db utero-ovarian preparations. The non-membrane receptor-bound elevation in insulin uptake by the tissues of db/db groups, relative to +/? samples (Fig. 7B), correlated with the elevated glucose utilization rates demonstrated by db/db utero-ovarian tissues relative to (+/?) groups (Fig. 7C). The P-HRx treatments promoted both insulin binding and glucose utilization in utero-ovarian samples from db/db groups. The enhanced glucose utilization occurred under persistent hyperglycemic, and moderated insulinemic, conditions (Fig. 3) in the db/db groups, with both oxidative metabolic parameters enhanced by P-HRx therapy (Table 1).

4. Discussion The results of the present studies define the capabilities of P-HRx to effectively moderate the severity of the hyperinsulinemic–hypercytolipidemic condition expressed in non-leptin influenced, C57BL/KsJ mice, by the diabetes (db/db) mutation under persistent systemic hyperglycemia.

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Fig. 5. Photomicrographs (1000×) of secondary, non-atretic ovarian follicles of control (+/?: A, C, E) and diabetic (db/db: B, D, F) 16-week-old C57BL/KsJ mice following either oil (A–D) or P-HRx (E and F) injections. High-resolution light micrographs (A and B) revealed the high density of interstitial (I), thecal (T) layer and follicular granulosa (G) cell lipid depositions observed in oil-HRx (db/db) groups (B) relative to controls (A). By DCSP, the normal distribution pattern and density of I and T lipid deposits were enhanced in both (+/?: C) and (db/db: D) groups. The effectiveness of P-HRx on reducing the G and T lipid deposit densities in diabetic (F) mice, to comparable P-HRx-treated (+/?: E) controls, was denoted by the remarkable reduction in total lipid accumulation as indicated by the reduced intensity of fluorescent yellow lipid pools compared to oil-HRx (db/db: D) mice. P-HRx did not disperse lipodepositions localized in I tissue compartments in (db/db) groups (F) to intensities noted in corresponding (+/?) mice (E).

Concurrently, the moderation of the phenotypic obesity index, as well as promotion of normalized utero-ovarian tissue weights coupled with suppression of the severe hypercytolipidemic organoatrophy which characterizes reproductive tract involution under the (db/db)-induced non-homeostatic endometabolic environment [2], indicates that chronic, low-dose P-HRx is an effective modulator of DOS influences on female reproductive tract involution. The moderating P-HRx influences on utero-ovarian cytopreservation were not through the correction of the persistent hyperglycemic state, a factor which has been recognized to be influenced by other steroidal or pre-steroidal synthesis pathway metabolites [8,9,16,17,20–22]. Instead, P-HRx effects occur through an apparent non-insulin membrane receptor influence [13], which enhances insulin-stimulated tissue glucose utilization rates [23], reducing db/db-stimulated lipogeneis

towards a more normal oxidative metabolic regulation of utero-ovarian glucometabolism. The resulting correction of glucose oxidative metabolism by P-HRx circumvents the stimulated glucose shunting towards lipodeposition in db/db mutants [2,7–9], thereby reducing tissue lipogenic activities and reducing the hypercytolipidemic compromise of normal utero-ovarian structure [2]. Of particular interest was the persistent hyperglycemic state noted in P-HRx groups, even under moderated systemic hyperinsulinemic conditions in 16-week-old mutants. These data suggest that while the P-HRx can influence the insulin-directed intra-cellular modulators of glucose metabolism [15,23] in utero-ovarian tissue types, that the systemic endometabolic imbalance associated with the DOS state continued to impose an abnormal hyperglycemic environment on other peripheral cell types, some of which are unresponsive to the noted


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Fig. 6. Influences of P-HRx on uterine and ovarian tissue lipase activities are represented as group (N = 3–6) mean values (±S.E.M.) for control (+/?) and diabetic (db/db) C57BL/KsJ mice at 16 weeks of age. (∗) Genotype-related significant (P ≤ 0.05) differences between (+/?) and (db/db) groups relative to treatment regime. Intra-group, treatment-related differences are denoted by (#). N.D.: non-detectable.

steroid-stimulated cellular metabolic counter-regulation of the non-homeostatic condition [16,22]. Thus, the cellular responsivity and glucometabolic-sensitivity to steroidal agents [24] must be recognized with respect to the evaluation of the tissue-specific, therapeutic efficacy of any metabolic agent capable of moderating the deleterious effects of the DOS state. Previous reports have indicated that other gender-dominant gonadal steroids have modulatory influences relative to the expressed severity of the DOS state in various species [6,9,11,25]. In males, pre-testosterone metabolites have proven to be effective modulators of the hyperglycemic component of the DOS (10–11), but have limited influences on the associated hyperinsulinemic-obesity components of the syndrome [12]. In addition, 17-B-estradiol (E) has been demonstrated to be a potent anti-hyperglycemic agent in females [8,9], but an ineffective modulator of hyperinsulinemia [6,9]. The findings from the current studies suggest that the noted independent, ovarian progesterone-estradiol influences [13], concurrently promoting both the P-modulating insulin release/cellular recognition properties [23], coupled with the E-normalization of the hyperglycemic metabolic environment [9], may explain the anti-diabetic efficacy noted for complexed oral contraceptive agents in humans [13]. The findings that cell-specific, insulin-modulating indices may be independently manipulated by P-therapy in a genetically mutant (db/db) diabetic-obese model, as compared to the hyperglycemic component of the DOS state which is responsive to E-sensitive glucometabolic

modulators [8,9], suggests that tissue-specific therapeutic endometabolic normalization of diabetes-associated female reproductive involution may be instituted, potentially promoting reproductive competency in support of the maintenance of utero-ovarian structural integrity [2]. Although female reproductive tract tissue has been used as an evaluation model in the current studies, previous studies focusing on steroidal modulation of glucometabolic indices in neural [20,21], hepatic [27], pancreatic [26], fat [22], and adrenal [22] tissue support the contentions of the present studies that steroid-sensitive peripheral tissue metabolism may be modulated under DOS conditions when tissue-specific sensitivity to steroidal agents is recognized. The noted modulation by P-HRx of utero-ovarian glucose utilization, tissue insulin binding, and cytoarchitectural indices in the present study, are not universally corrected parameters by steroidal therapy in all tissue types [8,22]. Although such steroid-responsive neural loci as the hypothalamus and limbic regions may be metabolically modulated and structurally preserved by exogenous HRx, cortical and isolated brainstem regions failed to demonstrate any endometabolic responsiveness to therapies which demonstrated protective influences from diabetes-induced premature neuronal degeneration [20,21]. In a similar manner, hepato-pancreatic tissues exhibit a species-, genderand steroid-specificity with respect to the efficacy of similar steroid-based therapies to moderate the influences of the DOS condition on cytostructural and metabolic parameters [8,9,13,26]. With the contribution of the present data identi-

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The promotion of normal cytostructure and function, even in genetically-mutant strains, suggests that therapeutic, steroidal modulation of intra-cellular metabolism may prove to be an effective counter-regulatory response to the chronic hyperglycemic state which characterizes all forms of expressed diabetes through cellular gluco- and lipo-toxicity [2,9,21]. In summary, the results of the current studies define and demonstrate that P-HRx is an effective modulator of DOS-stimulated, insulin-directed glucometabolic indices in the utero-ovarian tissue compartments of (db/db) C57BL/KsJ mutants. The ability of P-HRx to modulate phenotypic obesity, restore tissue weight indices, suppress the severity of the diabetes-induced hypercytolipidemia and lipogenic tissue metabolic parameters, while stimulating tissue glucose utilization, suggests that these counter-regulatory influences may be invoked through steroid-specific stimulation of homeostatic glucometabolic pathways capable of maintaining normal tissue integrity, even under persistent systemic hyperglycemic insult. Evaluation of the molecular mechanisms responsible for the counter-regulatory metabolic actions demonstrated by P-HRx therapies are currently in progress.

Acknowledgements The authors wish to express appreciation for the excellent endocytological assistance provided by Ms. Jessica Kueker.


Fig. 7. The influences of both the diabetes (db/db) mutation and P-HRx treatments on utero-ovarian insulin binding, insulin membrane receptor populations, and tissue glucose uptake rates are presented as group (N = 5–8 groups) mean values (±S.E.M.) for 16-week-old C57BL/KsJ mice. (∗) Significant (P ≤ 0.05) intergroup differences for each parameter for both oil- and P-HRx-treated groups.

fying the insulin-specific modulatory capabilities of P-HRx on utero-ovarian cytostructural preservation, continuing efforts focus on the integrative and co-modulatory influences of complexed steroidal therapies on metabolic-specific indices in an attempt to develop tissue-specific restoration treatments of abnormal cytoarchitectural and functional indices exposed to these non-homeostatic, DOS conditions.

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