The effect of diabetes and centrally administered insulin on anterior hypothalamic estrogen receptor alpha immunoreactivity.

Diabetic rats have characteristic reproductive deficits. Peripheral and central (intra-cerebro-ventricular [ICV]) insulin restores the reproductive phenotype to control levels. In this experiment, we evaluated a possible defect in steroid feedback by evaluating hypothalamic estrogen receptor (ER) alpha availability using ERalpha immunocytochemistry. Animals were ovariectomised and given estradiol and progesterone. Diabetic and control animals were given ICV insulin or saline. Nuclear and/or cytoplasmic ERalpha immunoreactivity was evaluated in the paraventricular nucleus (PVN) and the organum vasculosum laminae terminalis (OVLT). In the PVN, nuclear immunoreactivity was increased among diabetic, ICV insulin-treated animals. Diabetic saline-treated and nondiabetic animals had similar ERalpha immunoreactivity. The OVLT had lower numbers of immunoreactive neurons compared to the PVN; no differences among the treatment groups were found in the OVLT. Central insulin treatment increased the number of PVN nuclear ERalpha immunoreactive neurons among diabetic animals. However, there was no reduction in ERalpha when comparing saline-treated diabetic animals to non-diabetic rats, suggesting that decreases in hypothalamic ERalpha in the regions studied do not account for diabetes-induced reproductive deficits.

The effect of diabetes on alpha2-adrenergic receptor activity in the reproductive centers of the female rat brain.

OBJECTIVE: We hypothesized that known diabetes-induced deficits in female rat reproduction may result in part from decreased central alpha(2)-noradrenergic receptor density or affinity. STUDY DESIGN: Female rats were oophorectomized and divided into 2 groups; one group received streptozocin during the operation to induce diabetes, and the other served as a nondiabetic control group. Random blood glucose levels were measured. Half the rats in each group were killed on postoperative day 10, and half were killed on postoperative day 14. Direct radioligand binding assays were performed on tissue prepared from the hypothalamus, preoptic area, and cortex of each rat. Analysis of variance was used to evaluate intergroup differences in receptor concentration or equilibrium constant. RESULTS: We detected no significant difference in the mean receptor concentration or equilibrium constant between the groups with and without diabetes in the hypothalamus, the preoptic area, and the cortex on postoperative day 10 or 14. CONCLUSION: Diabetes-induced impairments in female rat reproduction do not involve alterations in alpha(2)-receptor density or affinity in the hypothalamus, preoptic area, or cortex.

Hormonal integration of neurochemical and sensory signals governing female reproductive behavior.

This review focuses on findings from our laboratory regarding mechanisms by which the ovarian steroid hormones, estradiol (E2) and progesterone (P), act in the hypothalamus (HYP) to regulate the expression of lordosis, an important component of female reproductive behavior in rats and many other species. The first section summarizes recent work suggesting that cGMP, perhaps via P-receptor activation, may be an intracellular mediator of the facilitatory actions of a variety of hormones and neurotransmitters on lordosis behavior in E2-primed rats. In the second section, we focus on E2 and P regulation of norepinephrine (NE) neurotransmission in the HYP. We review evidence that ovarian hormones act both peripherally and centrally to determine whether NE is released in the HYP in response to copulatory stimuli. We also suggest that the steroid milieu determines the cellular responses of hypothalamic neurons to released NE, favoring the activation of pathways implicated in the facilitation of both lordosis behavior and the preovulatory gonadotropin surge. It is likely that E2 and P have similar actions on other neurotransmitter and neuromodulator systems, thereby maximizing the probability that females are sexually receptive during the periovulatory period.

Intracerebroventricular leptin regulates hepatic but not peripheral glucose fluxes.

Acute intravenous infusions of leptin markedly alter hepatic glucose fluxes (Rossetti, L., Massillon, D., Barzilai, N., Vuguin, P., Chen, W., Hawkins, M., Wu, J., and Wang, J. (1997) J. Biol. Chem. 272, 27758-22763). Here we examine whether intracerebroventricular (ICV) leptin administration regulates peripheral and hepatic insulin action. Recombinant mouse leptin (n = 14; 0.02 or 1 microgram/kg.h) or vehicle (n = 9) were administered ICV for 6 h to conscious rats, and insulin action was determined by insulin (3 milliunits/kg.min) clamp and tracer dilution techniques. During physiologic hyperinsulinemia (approximately 65 microunits/ml), the rates of glucose uptake (Rd, 20.1 +/- 0.6 and 23.1 +/- 0.7 versus 21.7 +/- 0.6 mg/kg.min; p = NS), glycolysis and glycogen synthesis were similar in rats receiving low- and high-dose leptin versus vehicle. ICV leptin resulted in a 2-3-fold increase in hepatic phosphoenolpyruvate carboxykinase mRNA levels. Glycogenolysis and PEP-gluconeogenesis (2.1 +/- 0.3 mg/kg. min) contributed similarly to endogenous glucose production (GP) in the vehicle-infused group. However, gluconeogenesis accounted for approximately 80% of GP in both groups receiving ICV leptin, while hepatic glycogenolysis was markedly suppressed (0.7 +/- 0.3 and 1.2 +/- 0.3 versus 2.2 +/- 0.4 mg/kg.min, in rats receiving low- and high-dose leptin versus vehicle, respectively; p < 0.01). In summary, short-term ICV leptin administration: 1) failed to affect peripheral insulin action, but 2) induced a striking re-distribution of intrahepatic glucose fluxes. The latter effect largely reproduced that of leptin given systemically at much higher doses. Thus, the regulation of hepatic glucose fluxes by leptin is largely mediated via its central receptors.

Effects of diabetes and estradiol on norepinephrine release in female rat hypothalamus, preoptic area and cortex.

These studies determined whether diabetes and estradiol treatment altered norepinephrine (NE) release from hypothalamus, preoptic area (POA), and cortical slices from ovariectomized (OVX) female rats. Animals were sacrificed 12 days after the onset of streptozotocin-induced diabetes and 48 h following vehicle or estradiol injection. Brain slices were preloaded with 3H-NE, and release was evoked twice (S and S2) by electrical stimulation. Diabetes increased hypothalamic NE release during S1 regardless of the administration of vehicle or estradiol. Neither estradiol treatment nor diabetes alone affected NE release during S2 in the hypothalamus or POA. Estradiol treatment elevated NE release in the POA during S2 but only in diabetic animals. Moreover, estradiol elevated cortical NE release during S2 regardless of the presence or absence of disease. We also examined whether alpha2-adrenoceptor regulation of NE release was influenced by diabetes or hormone treatment. Enhancement of NE release by alpha2-adrenoceptor antagonism was evident in all 3 brain regions. However, alpha2-adrenoceptor regulation of NE release was unaffected by diabetes and hormone treatment. These findings suggest that diabetes alters NE release in the hypothalamus/POA of female rats. Additionally, this work identifies a novel action of estradiol to enhance stimulated NE release in the cortex of female rats.

Estradiol reduction of alpha 2-adrenoceptor binding in female rat cortex is correlated with decreases in alpha 2A/D-adrenoceptor messenger RNA.

We developed a quantitative RNAse protection assay for alpha 2A/D-adrenoceptor messenger RNA in rats to test the hypothesis that decreases in cortical alpha 2-adrenoceptor binding observed following estrogen treatment of ovariectomized rats correlate with reduced levels of messenger RNA encoding the predominant alpha 2-adrenoceptor subtype expressed in cortex. Estradiol treatment for 48 h reduced cortical alpha 2A/D-adrenoceptor messenger RNA by approximately 50% when compared to ovariectomized, oil-treated control animals. Estradiol down-regulation of alpha 2A/D-adrenoceptor messenger RNA was accompanied by a significant decrease in cortical alpha 2-adrenoceptor density, as measured by 3H-RX821002 binding. Estrogen treatment did not alter alpha 2A/D-adrenoceptor messenger RNA or alpha 2-adrenoceptor binding in female rat hypothalamus-preoptic area. This study provides the first evidence that estradiol regulates expression of postsynaptic alpha 2-adrenoceptors in female rat frontal cortex, suggesting a possible molecular substrate for hormonal modulation of cognitive function.

Deficits in reproductive behavior in diabetic female rats are due to hypoinsulinemia rather than hyperglycemia.

These studies determined whether deficits in reproductive behavior observed in streptozotocin (STZ)-induced diabetic female rats are caused by hyperglycemia or loss of insulin. Female Sprague-Dawley rats were ovariectomized and made diabetic by a single ip injection of STZ (75 mg/kg). Reproductive behavior was measured 12 days after the onset of hyperglycemia following the injection of estrogen and progesterone in doses known to restore reproductive behavior in nondiabetic rats. Rats in which STZ produced diabetes showed significantly reduced receptive and proceptive sexual behaviors. Normalization of blood glucose levels either by restricting diet or by phlorizin treatment failed to restore reproductive behavior in diabetic animals. However, even doses of insulin which were not fully effective in correcting peripheral hyperglycemia were able to prevent the STZ-induced behavioral deficit. No changes in general activity were observed in any experimental group as assessed by open field activity. The density of the norepinephrine transporter, as measured by [3H]nisoxetine binding, was reduced in the cortex but not in the brain stem, hypothalamus, or hippocampus of diabetic animals. Insulin treatment prevented the loss of cortical [3H]nisoxetine binding, and even partial normalization of blood glucose restored cortical [3H]nisoxetine binding to control levels. These findings suggest that diabetes-induced reproductive deficits are due to hypoinsulinemia and cannot be corrected simply by the normalization of blood glucose, whereas reductions in the density of cortical norepinephrine transporter result from hyperglycemia.

Estradiol regulation of alpha 1b-adrenoceptor mRNA in female rat hypothalamus-preoptic area.

Estradiol treatment for 48 h increases the density of alpha 1B-adrenoceptors in the hypothalamus-preoptic area of ovariectomized female rats by five- to six-fold. Present studies tested the hypothesis that estradiol elevation of hypothalamus-preoptic area alpha 1B-adrenoceptor density is correlated with increased levels of mRNA for this receptor. We developed a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for measuring brain alpha 1b-adrenoceptor mRNA. The primers chosen yielded the predicted 409 base pair PCR product when used to amplify authentic alpha 1b-adrenoceptor cDNA. The identity of the RT-PCR products from rat brain was confirmed by restriction digest analysis and sequencing. Moreover, there was a good correlation between the levels of alpha 1b-adrenoceptor mRNA measured by RT-PCR in liver, whole brain and cerebellum with previous measurements using Northern blots and RNAse protection assays. We then performed RT-PCR on total RNA from hypothalamic-preoptic area tissue taken from ovariectomized control rats and from ovariectomized rats injected once or twice with 2 micrograms of estradiol benzoate at 24 or 24 and 48 h before sacrifice. Exposure to estradiol for either 24 or 48 h significantly increased levels of alpha 1b-adrenoceptor mRNA by 86-110% in the hypothalamus-preoptic area of ovariectomized female rats when compared to oil-treated controls. We also examined whether estradiol regulates alpha 1b-adrenoceptor mRNA in the cortex. Cortical alpha 1b-adrenoceptor mRNA levels were reduced to approximately 20% of control levels when measured 24 h after hormone injection. A similar decrease in cortical alpha 1b-adrenoceptor mRNA was observed 48 h after estrogen administration. In summary, estradiol treatment significantly increases the level of alpha 1b-adrenoceptor mRNA in the hypothalamus-preoptic area, a brain region involved in the control of reproductive function. In the cortex, a brain region with relatively few estrogen receptors, the same estrogen treatment reduces alpha 1b-adrenoceptor mRNA levels.

Progesterone attenuation of alpha 1-adrenergic receptor stimulation of phosphoinositol hydrolysis in hypothalamus of estrogen-primed female rats.

These experiments examined whether the previously observed abolition by progesterone (P) of alpha 1-adrenergic potentiation of adenylyl cyclase activity in brain slices of estrogen-primed female rats is attributable to a reduced capacity of alpha 1-adrenoceptors to stimulate phosphoinositol hydrolysis. In preoptic area and hypothalamic slices from ovariectomized (OVX) female rats, both norepinephrine (NE) and the alpha 1-adrenergic agonist phenylephrine (PHE) were robust stimulators of inositol phosphate (IP) formation. The NE response was completely blocked by the alpha 1-adrenergic antagonist prazosin. PHE-induced IP formation in tissue from OVX females exposed for 48 h to estrogen alone was comparable to that in OVX controls. In hypothalamic tissue from OVX rats given estrogen plus P, NE and PHE activation of phosphoinositol hydrolysis was attenuated. Both chlorethylclonidine, an irreversible antagonist of alpha 1b-adrenoceptors, and 5-methylurapidil, an alpha 1a-selective antagonist, reduced NE-induced IP formation regardless of the hormonal condition of the animals. Analysis of PHE competition for [3H]prazosin binding indicated that agonist binding was of high affinity (Ki, 16-30 microM) and was unaffected by hormonal status. Therefore, P abolition of alpha 1-adrenergic augmentation of adenylyl cyclase is correlated with reduced phosphoinositol hydrolysis, but not with changes in alpha 1-adrenoceptor agonist binding affinity. Moreover, both alpha 1a- and alpha 1b-adrenoceptors appear to work together to stimulate this second messenger system in the female rat hypothalamus.

A thermodynamic analysis of estrogen regulation of alpha 2-adrenoceptor binding.

We previously demonstrated that in vivo estradiol treatment markedly attenuates alpha 2-adrenoceptor function and coupling to G-proteins in the hypothalamus of female rats. Ligand binding studies indicated that 48 h exposure to estradiol decreases the number of alpha 2-adrenergic receptors in the agonist high affinity state. In the present studies, when [3H]RX821002 was used to label brain alpha 2-adrenoceptors, the density of binding sites significantly increased in the hypothalamus and preoptic area 48 h after estrogen treatment. Moreover, the thermodynamics of ligand binding to alpha 2-adrenergic receptors in membranes of female rat hypothalamus were modified by the same estradiol treatments that reduce alpha 2-adrenoceptor function. In hypothalamic membranes from ovariectomized control rats, antagonist (RX821002)-receptor binding was primarily entropy-driven while agonist (oxymetazoline) binding had a higher enthalpy component. In membranes from estradiol-exposed animals, the entropic contribution to both agonist and antagonist bindings was markedly increased, and the enthalpy component was reduced. Since the thermodynamic characteristics of ligand-receptor binding are strongly correlated with efficacy in activating signal transduction [36], these data raise the intriguing possibility that steroids regulate transmembrane signaling by stabilization of a receptor conformation with reduced intrinsic efficacy.

Estradiol reduction of the agonist high affinity form of the alpha 2-adrenoceptor in the hypothalamus of female rats: identification as the alpha 2D subtype.

These studies examined which alpha 2-adrenoceptor subtype is expressed in the hypothalamus and preoptic area and the influence of estradiol administration on alpha 2-adrenoceptors in the hypothalamus of female rats. The alpha 2-adrenoceptor antagonist [3H] RX821002 bound to a single site in hypothalamus, preoptic area, and cortex membranes, with high affinity and low nonspecific binding, as determined by Scatchard and kinetic binding analyses. Competition for [3H]RX821002 binding in the hypothalamus and preoptic area by various noradrenergic agonists and antagonists revealed a unique pharmacological specificity with a high degree of similarity to that of the alpha 2D-adrenoceptor. Norepinephrine displacement of [3H]RX821002 binding in hypothalamic membranes from ovariectomized animals was monophasic and characterized by high affinity. In contrast, norepinephrine competition for [3H]RX821002 binding sites in the hypothalamus from rats exposed to estradiol for 48 hr was biphasic, and norepinephrine bound to both a high (18%) and a low (82%) affinity site in these membranes. Thus, the formation of agonist high affinity alpha 2D-adrenoceptor complexes was inhibited by prior exposure to estrogen. In both control and estradiol-exposed hypothalamic membranes, 100 microM 5'-guanylylimidodiphosphate [Gpp(NH)p] converted the norepinephrine competition curves to ones characterized by monophasic, low affinity binding. In addition, binding of the full alpha 2-adrenoceptor agonist [3H]UK-14,304 in the hypothalamus and preoptic area of female rats was concentration-dependently diminished by Gpp(NH)p treatment. Complete loss of [3H]UK-14,304 binding was effected by 100 microM Gpp(NH)p. This suggests that [3H]UK-14,304 may be useful in labeling the agonist high affinity state of alpha 2-adrenoceptors. Decreasing the incubation temperature in saturation studies from 25 degrees to 0 degrees increased [3H]UK-14,304 binding in hypothalamic membranes of control rats but not in membranes from estradiol-treated rats. Estradiol treatment for 48 hr decreased [3H]UK-14,304 binding in hypothalamic membranes by 34% (0 degrees) to 60% (25 degrees), without changing the Kd. These results suggest that the alpha 2D-adrenoceptor is the predominant subtype in the hypothalamus and preoptic area of female rats and that estradiol treatment markedly reduces the number of alpha 2D-adrenoceptors in the agonist high affinity state.

Estrogen regulation of noradrenergic signaling in the hypothalamus.

Hypothalamic circuits utilizing the monoamine neurotransmitter norepinephrine (NE) may be key elements upon which the ovarian steroids estradiol (E2) and progesterone (P) act to regulate female reproductive behavior. Recent studies have focused on the modulation of hypothalamic NE release by E2 and P treatments that facilitate sexual behavior. Brain microdialysis studies suggest that oxytocin, a neuropeptide known to enhance lordosis when infused into the ventromedial hypothalamus (VMH) of E2 + P-primed females, modulates NE release in the VMH. Systemic administration of oxytocin reliably enhances extracellular NE levels in the VMH of animals primed with moderate doses of both E2 and P. Thus, ovarian steroids may facilitate female sexual behavior in part by promoting oxytocin-induced NE release in the VMH. Studies examining the release of 3H-NE from superfused hypothalamic slices indicate that estrogen treatment also facilitates NE neurotransmission by attenuating alpha 2-adrenergic receptor-mediated inhibition of NE release. Hypothalamic alpha 2-adrenergic receptors are not downregulated by estrogen, suggesting that brain adrenoceptor function can be modulated by E2 independent of changes in receptor density. A model is proposed wherein E2 and P enhance hypothalamic NE release, leading to increased excitability of VMH neuronal activity and the expression of lordosis behavior.

Estradiol attenuates alpha 2-adrenoceptor-mediated inhibition of hypothalamic norepinephrine release.

These studies tested the hypothesis that estradiol facilitates norepinephrine (NE) neurotransmission by modulating alpha 2-adrenoceptor-mediated inhibition of NE release. KCl-induced overflow of 3H from superfused slices preloaded with 3H-NE was Ca2+ dependent. Hypothalamic slices from estradiol-treated rats exposed to a single KCl pulse (S1) had modestly (20%) but significantly elevated NE release when compared to slices from ovariectomized (OVX) rats. Blockade of alpha 2-adrenoceptors by pretreatment with the imidazoline antagonists idazoxan (IDA) and RX821002 (RX) markedly facilitated NE release during S1 in hypothalamic slices from OVX rats; this facilitation was attenuated or absent in slices from estradiol-treated rats. In additional studies slices were stimulated twice, 24 min apart (S1 and S2), for 3 min with 20 mM KCl. In the absence of drug, the amount of 3H-NE released during S2 was always less than the amount released during S1 (i.e., S2:S1 approximately 0.6), regardless of whether slices were from OVX or estradiol-treated females. When 10 microM IDA was applied after S1 and 15 min prior to S2, the S2: S1 ratio increased to 1.8 +/- 0.1 in hypothalamic slices from OVX animals. In contrast, the S2:S1 ratio rose only to 1.1 +/- 0.2 in slices from estradiol-treated animals. RX applied before S2 markedly increased the S2:S1 ratio in both hypothalamic and preoptic area slices from OVX rats but failed to increase the S2:S1 ratio in slices from estradiol-treated rats. Interestingly, the modest effects of alkaloid alpha 2-antagonists such as yohimbine and rauwolscine on NE release in hypothalamic and preoptic area slices were not modified by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol selectively regulates alpha 1B-noradrenergic receptors in the hypothalamus and preoptic area.

We previously demonstrated that estradiol administered in vivo elevates the number of alpha 1-adrenoceptors in preoptic area (POA) and hypothalamic membranes from ovariectomized female rats and potentiates alpha 1 receptor augmentation of beta-adrenoceptor-stimulated cAMP formation in slices from these brain regions. Present studies examined (1) if estradiol selectively regulates any alpha 1-adrenoceptor subtype, and (2) which alpha 1 receptor subtype mediates the augmentation of cAMP synthesis. Hypothalamic and POA membranes from estradiol-treated rats, when compared to ovariectomized rats, had modestly (30-50%) but significantly elevated numbers of 3H-prazosin (alpha 1) binding sites. Estradiol affected neither the number of alpha 1 receptor sites in frontal cortex nor the affinity of 3H-prazosin binding in any brain region examined. Results of binding studies conducted in the presence of chlorethylclonidine, a selective, irreversible inactivator of the alpha 1B receptor subtype, indicated that the estrogen-dependent increase in total alpha 1 binding sites in POA and hypothalamic membranes was attributable to a selective, five- to sixfold increase in alpha 1B receptor number. Progesterone had no measurable effects on alpha 1 receptor binding. Blockade of alpha 1B receptors with chlorethylclonidine eliminated phenylephrine augmentation of isoproterenol-stimulated cAMP formation in slices, whereas the alpha 1A antagonist 5-methyl-urapadil did not. This suggests that the alpha 1B receptor subtype potentiates cAMP formation. Thus, the increased alpha 1 receptor augmentation of cAMP formation seen in slices from estradiol-treated rats is correlated with increased alpha 1B receptor number.

Estradiol regulates the number of ?(1) but not ? or ?(2) noradrenergic receptors in hypothalamus of female rats.

Present experiments examined whether previously observed hormone-dependent differences in norepinephrine-stimulated cAMP accumulation in hypothalamic and preoptic area slices are attributable to differences in noradrenergic receptor number or binding affinity. When compared to ovariectomized controls, hypothalamic and preoptic area membranes from estradiol-treated rats had significantly elevated numbers of [(3)H]prazosin (?(1)) binding sites. Estradiol affected neither the number of ?(1) sites in frontal cortex nor the affinity of [(3)H]prazosin binding in any brain region sampled. Estradiol had no effect on [(3)H]idazoxan (?(2)) or [(3)H]dihydroalprenolol (?) binding in hypothalamus, preoptic area or cortex. Progesterone reversed estradiol elevation of prazosin binding in preoptic area but had no other measurable effects on any noradrenergic receptor binding when given alone or in combination with estradiol. Neither estradiol nor progesterone altered binding of radiolabeled antagonists when they were included in the in vitro incubation mixture. These data suggest that the increased ?(1) receptor augmentation of cAMP accumulation seen in hypothalamic and preoptic area slices from estradiol-treated rats is correlated with increased ?(1) receptor number. In contrast, estradiol attenuation of ? receptor function and progesterone depression of norepinephrine-stimulated cAMP generation in slices from estradiol-treated females is not correlated with downregulation of any noradrenergic receptor subtype.