Effects of dexamethasone in vivo and in vitro on hexose transport in brain microvasculature.

Glucocorticoids induce hyperinsulinemia, hyperglycemia, and depress glucose transport by aortic endothelium. High glucocorticoid doses are used for many diseases, but with unknown effects on brain glucose transport or metabolism. This study tested the hypothesis that glucocorticoids affect glucose transport or metabolism by brain microvascular endothelium. Male rats received dexamethasone (DEX) s.c. with sucrose feeding for up to seven days. Cerebral microvessels from rats treated with DEX/sucrose demonstrated increased GLUT1 and brain glucose extraction compared to controls. Glucose transport in vivo correlated with hyperinsulinemia. Pre-treatment with low doses of streptozotocin blunted hyperinsulinemia and prevented increased glucose extraction induced by DEX. In contrast, isolated brain microvessels exposed to DEX in vitro demonstrated suppression of 2-deoxyglucose uptake and glucose oxidation. We conclude that DEX/sucrose treatment in vivo increases blood-brain glucose transport in a manner that requires the effects of chronic hyperinsulinemia. These effects override any direct inhibitory effects of either hyperglycemia or DEX.

Conversion of estrogen receptor from a state with low affinity for estradiol into a state of higher affinity does not require 4S to 5S dimerization.

This study was undertaken to establish whether the heat-promoted conversion of receptor-estradiol complex (RE2) from a state with fast into a state with slow E2 dissociation requires 8S/4S to 5S transformation. The calf uterine estrogen receptor labeled with [3H]E2 at 0 C (state with low affinity for E2) was immobilized on hydroxylapatite (HAP) in the absence (8S oligomer) or presence (4S monomer) of 0.4 M KCl and heated at 28 C in the presence of unlabeled diethylstilbestrol. Under these conditions, the dissociation of [3H]E2 was biphasic and occurred at rates similar to those obtained with R[3H]E2 free in cytosol. In contrast to the latter, however, the heat-promoted conversion of HAP-immobilized R[3H]E2 into a state of higher affinity for E2 was not accompanied by receptor dimerization, since the HAP eluate (0.4 M phosphate buffer) contained only the 4S monomer; upon reheating or desalting of this eluate, 4S to 5S dimerization occurred. The heat-promoted formation of 4S RE2 monomers with higher affinity for E2 was not due to monomer-HAP interactions, since even after elution from HAP, the 4S RE2 remained in the state of higher affinity for E2. Addition of pyridoxal 5'-phosphate to slow dissociating, high affinity 5S R[3H]E2 dimers free in cytosol induced rapid [3H]E2 dissociation, although the receptor remained unaltered in the transformed dimerized state. The effect of PLP was readily reversed by the addition of lysine. It is proposed that the 4S receptor monomers exist in two conformational states; upon E2 binding to the low affinity state, conformational changes result in stronger interactions between the steroid and the amino acid residues of the estrogen-binding domain; thus, the rate of E2 dissociation decreases. The formation of this 4S RE2 state with higher affinity for E2 is independent from receptor dimerization. A model is presented in which 4S to 5S transformation may lead to stabilization of 4S monomers in the conformation with higher affinity for E2.

Estriol and estradiol interactions with the estrogen receptor in vivo and in vitro.

The cytosolic estrogen receptor (calf uterus) bound to estradiol (E2) at 0 degrees C changes from a state with fast into a state with slow E2 dissociation rates when placed at 28 degrees C. This temperature accelerated transition in receptor affinity for its ligand takes place within 10 min at 28 degrees C. Similarly, receptor bound to estriol (E3) at 0 degrees C changes, when heated, from a state with fast into a state with slow E3 dissociation. The main difference between RE2 and RE3 was that E3 dissociates from unheated 8S RE3 and heat-transformed 5S RE3 at a much faster rate than E2 from RE2 . In the mature ovariectomized rat a slow dissociating 5S receptor estrogen complex is found in nuclei 1 h after injection of [3H]E2 or [3H]E3. In vitro dissociation of these 2 estrogens from this nuclear bound receptor formed in vivo takes place at rates similar to those from heat-transformed cytosolic RE2 or RE3 complexes. Addition of pyridoxal 5'-phosphate (PLP) to the slow-dissociating heat-transformed 5S estrogen receptor complexes causes rapid dissociation of E2 or E3; this effect is dose-dependent and is not due to disruption of 5S dimers, since after PLP addition RE2 and RE3 sediment unchanged as 5S dimers. The presence of a large excess of non-radioactive 4S RE3 does not interfere with the temperature induced rapid transition of 4S R[3H]E2 complexes from the state with fast into a state with slow E2 dissociation kinetics. A model is presented to explain the temperature induced biphasic estrogen dissociation from the receptor. It is proposed that the low affinity 4S RE2 monomer undergoes a temperature and estrogen dependent conformation change, such that the ligand is "locked" into the receptor's binding site. This conformational change results in the formation of a high affinity 4S monomer from which estrogen dissociates at a slower rate. This reaction is independent from subsequent 4S to 5S dimerization (transformation). The different rates of ligand dissociation from the low and high affinity 4S receptors reflect the different interactions (hydrophobic and hydrogen bonding) of E2 and E3 with the estrogen binding domain.