In the corpus luteum, prostaglandin F2 alpha (PGF2 alpha) appears to be a physiological agent with both antisteroidogenic and luteolytic actions. It is hypothesized that the antisteroidogenic action of PGF2 alpha acts through altered transport of cholesterol to the mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc). However, the effect of PGF2 alpha on the expression of the putative cholesterol transport protein, sterol carrier protein-2 (SCP2; 13.2 kilodaltons), has not been examined. In this study, the decline in serum progesterone after PGF2 alpha injection was examined in parallel with altered ovarian SCP2, P450scc, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) protein and messenger RNA (mRNA) levels. Rats (28 days old) were treated with 8 IU PMSG to induce follicular development and ovulation. Ten days after ovulation, animals were treated with PGF2 alpha (single or multiple injections; 100-250 micrograms each) or left untreated. Ovarian SCP2, P450scc, and 3 beta HSD protein and mRNA levels were examined 0 (time zero), 4, and 8 h post-PGF2 alpha treatment using Western and Northern blot analysis. SCP2 mRNA levels were also examined using a highly sensitive ribonuclease protection assay that detects a 429-base pair SCP2-mRNA specific sequence. The results indicate that serum progesterone was significantly reduced 4 and 8 h after PGF2 alpha injections (P < 0.001; n = 6/time point). The decline in progesterone paralleled a 50-60% reduction in 3 beta HSD protein and mRNA levels by 4 h post-PGF2 alpha. Protein and mRNA levels for 3 beta HSD returned to control values by 8 h post-PGF2 alpha treatment. P450scc expression was also reduced at 4 h (44-54%), but by 8 h, both protein and mRNA levels had increased above the normal control levels (P < 0.02). In contrast, the 0.8-kilobase SCP2-specific mRNA transcript was reduced to 50% and 80% of the pre-PGF2 alpha treatment level at 4 and 8 h, respectively (P < 0.01). SCP2 ribonuclease protection assay analysis also indicated that SCP2 mRNA levels were reduced 65% (P < 0.03) and 85% (P < 0.01) by 4 and 8 h post-PGF2 alpha treatment compared to those in time zero ovarian tissue. Consistent with the loss of SCP2 mRNA expression, Western blot analysis indicated that a 15-kilodalton SCP2-immunoreactive protein (presumably the pro-SCP2 form) was significantly reduced or absent in the PGF2 alpha treated animals (P < 0.04).(ABSTRACT TRUNCATED AT 400 WORDS)

While a strong relationship between the hypercholesterolemia of diabetes and premature atherosclerosis is established, the etiology for the elevation in serum cholesterol in this disease is unknown. To determine whether diabetic hypercholesterolemia may be related to alterations in hepatic cholesterol transport capacity, sterol carrier protein-2 (SCP2) expression was examined in rats treated with streptozotocin (SZT). Furthermore, this study examined whether 17β-estradiol and insulin confer a protective effect on liver cholesterol homeostasis by maintaining hepatic SCP2 levels. SCP2 protein and mRNA expression were examined 13 days following SZT-induced diabetes onset and in diabetic rats treated with estradiol (1 cm silastic implant) or insulin (12 units/day). Data indicate that SCP2 protein levels were significantly reduced in the diabetic animals and that SCP2 protein expression in the liver was inversely related to the level of serum cholesterol in the diabetic animals. In contrast, SCP2 mRNA levels examined by slot blot, ribonuclease protection assay, and Northern blot analysis were significantly elevated. Both insulin and estradiol were able to enhance the expression of SCP2 protein in the liver following SZT treatment. The results of this investigation clearly indicate that hepatic SCP2 protein levels are significantly altered in the diabetic state suggesting that cholesterol transport capacity is reduced in the SZT-treated diabetic rat. The inverse relationship between serum cholesterol and hepatic SCP2 protein content suggests that the reduction in this protein may be a contributing factor in diabetic hypercholesterolemia.

Reproductive dysfunction in the diabetic female rat is associated with impaired folliculogenesis, reduced corpus luteum progesterone output, and spontaneous abortion. The underlying mechanism for reduced steroid production remains unresolved. In this study we examined whether or not diabetes alters levels of P450 side-chain cleavage enzyme (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), or the cholesterol transport proteins, steroidogenic acute regulatory (StAR) protein and sterol carrier protein-2 (SCP2), leading to lower progesterone levels and pregnancy loss. Rats (Day 3 pregnant) received an injection of streptozotocin (STZ, 60 mg/kg; i.v.) to induce a diabetic state; P450scc, 3 beta-HSD, and SCP2 were examined by Western and Northern blot analysis in ovarian tissue 12 days after injection of STZ (diabetic rats, n = 12) or vehicle (nondiabetic rats, n = 12). Serum progesterone, triglyceride, and beta-hydroxybutyrate (beta-HBA) levels were also examined. Results indicate that diabetic rats that aborted (diabetic-fetus [Ft], n = 6) had significantly lower progesterone levels (7.04 +/- 2.6 ng/ml; p < 0.004) than nondiabetic animals (108.6 +/- 5.15 ng/ml) and diabetic +Ft animals (74.3 +/- 8.9 ng/ml, n = 6). Western blot analysis of ovarian P450scc and 3 beta-HSD in the nondiabetic rats and the diabetic rats with fetuses indicated no significant difference. In contrast, ovaries from diabetic animals without fetuses had significantly lower SCP2 levels (p < 0.017) compared to controls. Concomitant with the reduction in SCP2, a 58-kDa SCP2-immunoreactive protein, referred to as sterol carrier protein-X (SCPx), increased significantly (p < 0.001). The C-terminal sequence of SCPx is identical to SCP2, while its N-terminal region is homologous with 3-oxoacyl coenzyme A thiolase, an enzyme involved in fatty acid metabolism. Increased SCPx expression coincided with increased serum triglyceride and beta-HBA levels, suggesting that the enhanced SCPx level may coincide with an ovarian shift to fatty acid metabolism. When SCPx steady-state mRNA levels were measured using an SCPx-specific riboprobe (280-bp protected fragment) in a ribonuclease protection assay, ovarian SCPx mRNA levels in the diabetic animals were increased 4.2-fold compared to control SCPx mRNA levels. Ovarian StAR mRNA levels were increased slightly in the diabetic animals, and ovarian P450scc and 3 beta-HSD mRNA levels were increased 3-fold in the diabetic animals that aborted relative to the nondiabetic animals and the +Ft diabetic animals. Results of this study confirm that SCPx mRNA levels are elevated following diabetes onset and that StAR, P450scc, and 3 beta-HSD mRNA levels do not correspond with the reduced steroid hormone profile associated with diabetes. These results are concordant with the possibility that reduced steroid levels in the diabetic animals reflect a loss of SCP2-mediated cholesterol transport capacity as SCPx/3-oxoacyl coenzyme A thiolase expression is enhanced.