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Effects of estradiol and progesterone on diabetes-associated utero-ovarian atrophy in C57BLKsJ dbdb mutant mice.

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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.
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