Carboxy-terminal deletion of the HDL receptor reduces receptor levels in liver and steroidogenic tissues, induces hypercholesterolemia, and causes fatal heart disease
Rinku PalQingen KeGermán PihánAyce YesilaltayMarsha PenmanLi WangChandramohan ChitrajuPeter M. KangMonty KriegerOlivier Kocher
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The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.Keywords:
Reticulocytosis
In the sheep, in contrast to the rat, arginine vasopressin (AVP) is a more potent stimulus to ACTH secretion from the anterior pituitary (AP) than CRF. To further explore this difference, we have compared [3H]AVP and [125I]-[Nle21 Tyr32] ovine CRF binding in membranes prepared from rat and sheep AP. Between species, no difference in affinity of binding was found for either ligand. In contrast, the concentration of AVP receptors in sheep AP was twice that in rat, whereas that of CRF receptors was only one tenth. AVP receptor concentration in sheep AP was not altered by chronic (10 day) dexamethasone administration, but fell to 60% of control after chronic (60 day) hypothalamo-pituitary-disconnection. The increased level of AVP receptors and the much lower level of CRF receptors in sheep compared with rat may thus provide an explanation for our previous findings of increased sensitivity to AVP and a very poor response to CRF in stimulating ACTH release from the sheep AP. In addition the finding that AVP receptor numbers are reduced in the hypothalamo-pituitary-disconnected sheep suggests that hypothalamic factors may play a role in regulating AVP receptor concentration in the ovine AP gland. (Endocrinology127: 2085–2089, 1990)
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The pituitary regulation of the sexually differentiated PRL receptor in rat liver was studied. PRL receptors were measured in a crude membrane fraction (105,000 × g pellet) using [125I]iodohuman PRL as tracer. Human GH (hGH), continuously administered by Alzet osmotic minipumps with an infusion rate of 5 μg/h for 1 week, was shown to induce PRL receptors in livers from male and female hypophysectomized-gonadectomized rats. The PRL receptors were increased to a level found in control female rat livers. This inductive effect of hGH was also seen in adrenalectomized and thyroidectomized male rats. In intact male rats given hGH, PRL receptors were increased to a female level in 4–7 days. hGH was effective in doses of 2.5 and 5 Μg/μl in inducing a female receptor pattern. The induced PRL receptors in male rats had characteristics similar to those of hepatic PRL receptors in female rats when data were calculated according to Scatchard (Kd = 0.13 × 10-9vs. 0.15 × 10-9 M; number of binding sites 88 vs. 57 fmol/mg protein). Also, the endogenous rat hormones, rat PRL (rPRL) and rat GH (rGH), were administered by minipumps to hypophysectomized male rats. With the infusion rate used (10 μg/h), rPRL had no effect, whereas rGH (NIAMDD-B6) increased PRL receptor levels to approximately 37% of the female control level. A more complete induction of PRL receptors (75% of the female control levels) in hypophysectomized males was achieved using another preparation of rGH (NIAMDD 1-4). Also, in hypophysectomized female rats, rPRL was ineffective in inducing PRL receptors. On the other hand, ovine PRL was found to give a partial restoration of PRL receptors in hypophysectomized female rats. The results indicate that GH or a peptide related to GH may be involved in the regulation of hepatic PRL receptors. However, the results do not rule out the possibility that rPRL, when present in doses other than those used in the present investigation, may also play a role in receptor regulation.
Hypophysectomy
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Corticosterone
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In vivo administration of gonadotropin-releasinghormone (GnRH) inhibits testosterone production in intact rats and increases testicular GnRH receptors. To delineate the relationship of these effects and to determine whether GnRH induced its testicular receptor by an extrapituitary mechanism, GnRH and LH receptors were studied in intact or hypophysectomized (H) rats. Adult, H, or sham-operated (S) males were injected with GnRH (6.7 μg every 8 h for 1, 2, 4, and 6 days). Some H rats were also treated with GH and PRL (150 μg/day) to maintain LH receptors. Decapsulated testes were assayed for GnRH and LH receptors or incubated to assess testosterone production in the presence or absence of hCG. GnRH treatment in S rats resulted in a biphasic change in GnRH receptor numbers. The basal receptor concentration (212 ± 27 fmol /mg protein) increased to a peak at 2 days (501 ± 32 fmol/mg) before declining to control values after 6 days of treatment. GnRH receptors increased 2-fold 1 day after hypophysectomy (449 ± 52 fmol/mg) and were further elevated by GnRH treatment (718 ± 49 fmol/mg). Receptor concentrations remained at that level throughout the 6 days of GnRH treatment. GH and PRL administration to H rats partially inhibited the rise in GnRH receptors after hypophysectomy. This hormone treatment did not alter the GnRH-induced increase in receptors after 1 or 2 days, but decreased the response on days 4 and 6 by 63%. hCG-stimulated testosterone production was reduced by 50% on all days of GnRH treatment in S rats, while LH receptors were unchanged on the first day, but were reduced by 60% on days 2–6 of treatment. In contrast, testosterone production was only marginally inhibited after 6 days of GnRH treatment in both H and H plus GH- and PRL-treated animals, while LH s receptors were not different from control values. However, when steroidogenic responsiveness in H animals was maintained with GH, PRL, and 5 μg/day LH, testosterone production was inhibited to the same degree as in S animals after 6 days. These results indicate that GnRH can directly increase GnRH testicular receptors, but the response is modulated by anterior pituitary hormones. The decrease in LH receptors in S rats appears to be a result of increased LH secretion and not a direct effect of GnRH at the dose used. Finally, the inhibitory effect of GnRH on testosterone production is temporally dissociated from the induction of GnRH receptors and the down-regulation of LH receptors, suggesting that it is a postreceptor-mediated event.
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The role of vasopressin (VP) in the regulation of pituitary corticotropin-releasing factor (CRF) receptors was studied by examining the effects of adrenalectomy and VP infusion on pituitary CRF receptors in genetically VP-deficient rats (di/di) and Long-Evans control rats. Binding studies with [125I]Tyr-ovine CRF in 30,000 × g anterior pituitary membranerich fractions revealed similar characteristics for the CRF receptors in Long-Evans and di/di rats, with Kd values of 2.4 ± 0.6 and 1.9 ± 0.2 nM, respectively, and receptor concentrations of 278 ± 31 and 286 ± 43 fmol/mg, respectively. Two days after adrenalectomy, the pituitary CRF receptor concentration decreased by 72 ± 4.2% in Long-Evans rats, but by only 20.3 ± 5.6% in di/di rats. CRF receptor affinity was unchanged after adrenalectomy (Kd = 1.7 ± 0.5 nM; n = 8). To determine whether VP deficiency is responsible for the smaller decrease in CRF receptor in di/di rats, the effect of exogenous VP infusion (100 ng/min) by sc osmotic minipumps was studied in adrenalectomized di/di rats. Two days after adrenalectomy, pituitary CRF receptors were reduced by 21 ± 8% in control di/di rats, whereas a 77.7 ± 1.8% decrease was observed in VP-infused di/di rats, comparable to the effect of adrenalectomy in Long-Evans rats. VP infusion also caused a significant 35 ± 2% decrease in CRF receptors in the pituitaries of sham-operated di/di rats, with no change in CRF receptor affinity. In Sprague-Dawley rats, VP or CRF infusion (100 ng/min) decreased pituitary CRF receptors by 14 ± 1.9% and 46 ± 3%, respectively. However, the combined infusion of both peptides caused a 65% ± 4.2 decrease, similar to that observed after adrenalectomy. In vitro incubation of quartered pituitaries with VP or CRF for 4 h reduced CRF receptors by 23.1 ± 8.2% and 38.2 ± 3.8%, respectively, while simultaneous preincubation with both peptides was followed by a decrease of 55.3 ± 5.3%. These findings indicate that increased hypothalamic release of VP contributes to the down-regulation of pituitary CRF receptors after adrenalectomy. (Endocrinology121: 2093–2098, 1987)
Corticotropin-releasing hormone
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To evaluate the capacity of the myocardium in aged rats to respond to hyperthyroidism, we quantified myocardial β-adrenergic receptors in female Fischer 344 rats of 3, 12, and 24 months of age. In T3-treated rats (500 μg T3/kg·day for 3 days), myocardial β-adrenergic receptors, as measured by [3H]dihydroalprenolol binding, were significantly increased (P < 0.01) over controls in 3-, 12-, and 24-month-old animals. The data demonstrate that senescent rats retain the capacity to increase myocardial β-adrenergic receptors in response to exogenous hyperthyroidism. In the myocardium, the mechanism of decreased catecholamine responsiveness in aging appears to be at other than the β-adrenergic receptor site, since receptor density is unaltered with age, as is receptor modulation in response to hyperthyroidism.
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The effects of hCG and various pituitary hormones on type I insulin-like growth factor (IGF) receptors of purified Leydig cells of hypophysectomized rats were studied. The number of type I IGF receptors of Leydig cells obtained from hypophysectomized rats (18.0 ±1.5 fmol/106 cells) was lower than that in normal rats (54.6 ± 5.3 fmol/106 cells; P < 0.05). After a single administration of hCG (10 U, ip), specific binding of [125I]IGF-I to purified Leydig cells increased 3-fold. Scatchard analyses of the binding data suggested that increased binding was the result of an increase in receptor number, whereas binding affinity remained unaltered. Type I IGF receptor increased within 12 h and remained persistently elevated 96 h after hCG treatment. Administration of hCG (10 U, ip) daily for 5 days increased type I IGF receptor levels to 73.2 ± 8 fmol/ 106 cells (P < 0.001). FSH caused a small but significant increase in type I IGF receptors. Concomitant administration of FSH and hCG further enhanced IGF-I-binding capacity. IGF-I-binding affinity of Leydig cells treated with FSH or FSH plus LH was not significantly different from that in the control hypophysectomized rats. Daily administration of GH for 5 days also upregulated type I IGF receptors, whereas PRL had no effect. FSH, GH, and PRL administration had no effect on serum testosterone levels. Serum testosterone levels increased to 3.99 ± 0.35 ng/ml after 5 days of treatment with hCG. Concomitant administration of FSH and hCG caused a further increased in serum testosterone levels (6.13 ± 0.46 ng/ml; P < 0.01). The present study suggests that type I IGF receptors of Leydig cells can be up-regulated by LH, FSH, and GH. However, hCG/LH seems to be the most important factor in maintaining and regulating type I IGF receptors of Leydig cells. Steroidogenic and growthpromoting effects of hCG and pituitary hormones on Leydig cells may be mediated by increased type I IGF receptors. (Endocrinology123: 134–139,1988)
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The specific binding of [125I]epidermal growth factor ([126I]EGF) to hepatic microsomal membranes was about 2-fold higher in adult male than in adult female rats. Scatchard analysis of the binding data showed that the sex difference in EGF binding was due to the difference in EGF receptor concentration rather than to a change in receptor affinity. From the developmental study, an apparent sex difference in EGF binding was observed from the pubertal period (4 weeks of age). Castration of adult male rats slightly, but significantly, decreased the EGF receptor level; and moreover, treatment of adult females with testosterone increased it only slightly. On the other hand, castration of neonatal male rats decreased the EGF receptor content almost to the female level. The decreased level of the receptor was completely restored by the combination of neonatal and pubertal treatments with testosterone. Neonatal or pubertal treatment alone of castrated animals had no significant effect on the decreased level of EGF receptors. These effects of testosterone were similarly observed when normal female rats were treated with the steroid. Moreover, hypophysectomy of the rats resulted in the marked decrease in EGF receptors only in the male animals. Treatment of hypophysectomized rats with either testosterone or T3 had no apparent effect on the EGF receptors. The membrane protein, cross-linked with [125I]EGF, had a mol wt of 170,000, and this protein (EGF receptor) was phosphorylated basally or by the addition of EGF. The rate of affinity labeling, or phosphorylation of EGF receptors, was in good agreement with the results of the EGF binding study. These results strongly suggest that the EGF receptor level in rat liver plasma membranes is in part regulated by the hypothalamopituitary unit and that neonatal androgens are essential for this regulation, probably through their effects on the hypothalamus. (Endocrinology122: 1707–1714, 1988)
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ABSTRACT A single injection of gonadotrophin-releasing hormone (GnRH) (60 ng s.c., 42·9 nmol) induced biphasic GnRH receptor regulation in normal intact adult female mice. A transient 22% receptor decrease occurred 30–60 min after injection of GnRH when peak serum decapeptide concentrations were reached (137 ± 41 ( s.e.m. ) ng/l). This GnRH receptor decrease occurred shortly after the peak serum LH values at 15–30 min. The subsequent rapid (within 1 h) return of GnRH receptor levels to normal suggested transient receptor occupancy by GnRH rather than true receptor loss. At 8 h after injection of GnRH a significant 35% increase in GnRH receptors was consistently observed, when serum GnRH levels were undetectable and serum LH had returned to basal levels. This receptor increase was not due to increased receptor affinity, and was prevented by a non-specific protein synthesis inhibitor. Ovariectomy, which caused a 50% fall in GnRH receptors (59·4 ± 4·9 fmol/pituitary gland in intact controls; 26·9 ± 2·6 in ovariectomized mice) abolished the induction by GnRH of its own receptors, although the initial transient decrease occurred over the period of the acute serum LH and FSH rise. Despite a 50% reduction in GnRH receptors in ovariectomized mice, increased serum gonadotrophin levels and responsiveness to GnRH were maintained, indicating dissociation between receptor changes and gonadotrophin levels. No GnRH receptor up-regulation was observed 8 h after a single GnRH injection (60 ng s.c.) in either intact or orchidectomized normal male mice. However, the same treatment doubled GnRH receptors in GnRH-deficient ( hpg ) female mice. While GnRH appears to up-regulate its own receptors by a direct action on pituitary gonadotrophs in the GnRH-deficient mouse its action in the normal female mouse pituitary appears secondary to stimulation of a gonadal product, presumably oestrogens. J. Endocr. (1985) 107, 41–47
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