HIP-70: a protein induced by estrogen in the brain and LH-RH in the pituitary.

Estrogen and luteinizing hormone-releasing hormone (LH-RH) interact to influence both behavior and gonadotropin release. However, little is known about the biochemical mechanisms that mediate the effects of these hormones or their interactions. The most prominent protein induced by estrogen in the ventromedial hypothalamus has the same amino-terminal sequence as the most prominent protein induced by LH-RH in the pituitary in vitro and in vivo; these proteins comigrate on two-dimensional gels. Furthermore, the hormonal induction may be caused by modification of a constitutive protein with the same molecular weight (70,000) but a slightly more acidic isoelectric point, whose level is inversely related to the level of the induced form after estrogen treatment. Thus estrogen and LH-RH may interact by additively or synergistically inducing this protein, which is called HIP-70.

Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Ovarian and steroidal influences on neuroendocrine aging processes in female rodents.

Some Mammalian aging processes involve effects of steroids on the brain and pituitary. An ovary-dependent, neuroendocrine aging syndrome of laboratory rats and mice is described in this article. This syndrome can be attenuated during aging by chronic ovariectomy and can be prematurely induced in young rodents by sustained exposure to estradiol (E2). The limited follicular stock in the ovary is proposed to be a major pacemaker of aging in this neuroendocrine syndrome; ovarian aging may interact with neuroendocrine aging. Ovary-independent neuroendocrine changes occur as well. We also discuss developmental influences on adult aging in rodents and other examples in which adult lower mammals are sensitive to long lasting effects of steroids on the brain and pituitary. Possible molecular mechanisms are considered. In view of the long lasting effects of E2 and other steroids on lower mammals, the potential for long term effects of ovarian steroids on the human brain and pituitary warrants continued evaluation.

Hypothalamic glucose sensor: similarities to and differences from pancreatic beta-cell mechanisms.

Glucose-responsive neurons in the ventromedial hypothalamus (VMH) are stimulated when glucose increases from 5 to 20 mmol/l and are thought to play an essential role in regulating metabolism. The present studies examined the role of glucose metabolism in the mechanism by which glucose-responsive neurons sense glucose. The pancreatic, but not hepatic, form of glucokinase was expressed in the VMH, but not cerebral cortex, of adult rats. In brain slice preparations, the transition from 5 to 20 mmol/l glucose stimulated approximately 17% of the neurons (as determined by single-cell extracellular recording) from VMH but none in cortex. In contrast, most cells in both VMH and cortex were silent below 1 mmol/l and active at 5 mmol/l glucose. Glucosamine, 2-deoxyglucose, phloridzin, and iodoacetic acid blocked the activation of glucose-responsive neurons by the transition from 5 to 20 mmol/l glucose. Adding 15 mmol/l mannose, galactose, glyceraldehyde, glycerol, and lactate to 5 mmol/l glucose stimulated glucose-responsive neurons. In contrast, adding 15 mmol/l pyruvate to 5 mmol/l glucose failed to activate glucose-responsive neurons, although pyruvate added to 0 mmol/l glucose permitted neurons to maintain activity. Tolbutamide activated glucose-responsive neurons; however, diazoxide only blocked the effect of glucose in a minority of neurons. These data suggest that glucose-responsive neurons sense glucose through glycolysis using a mechanism similar to the mechanism of pancreatic beta-cells, except that glucose-responsive neurons are stimulated by glycerol and lactate, and diazoxide does not generally block the effect of glucose.

Cerulenin mimics effects of leptin on metabolic rate, food intake, and body weight independent of the melanocortin system, but unlike leptin, cerulenin fails to block neuroendocrine effects of fasting.

Cerulenin and a related compound, C75, have recently been reported to reduce food intake and body weight independent of leptin through a mechanism hypothesized, like leptin, to involve hypothalamic nutrition-sensitive neurons. To assess whether these inhibitors act through mechanisms similar to mechanisms engaged by leptin, ob/ob and Ay (agouti) mice, as well as fed and fasted wild-type mice, were treated with cerulenin. Like leptin, cerulenin reduced body weight and food intake and increased metabolic rate in ob/ob mice, and cerulenin produced the same effects in wild-type mice, whereas lithium chloride, at doses that produce conditioned taste aversion, reduced metabolic rate. However, in contrast to leptin, cerulenin did not prevent effects of fasting on plasma corticosterone or hypothalamic levels of neuropeptide Y, agouti-related peptide, pro-opiomelanocortin, or cocaine- and amphetamine-related peptide mRNA. Also, in contrast to leptin, cerulenin was highly effective to reduce body weight in Ay mice, in which obesity is caused by blockade of the melanocortin receptor. These data demonstrate that cerulenin produces metabolic effects similar to effects of leptin, but through mechanisms that are independent of, or down-stream from, both leptin and melanocortin receptors.

Adrenalectomy reverses obese phenotype and restores hypothalamic melanocortin tone in leptin-deficient ob/ob mice.

In genetically obese leptin-deficient ob/ob mice, adrenalectomy reverses or attenuates the obese phenotype. Relative to lean controls, ob/ob mice also exhibit decreased hypothalamic proopiomelanocortin (POMC) mRNA and increased hypothalamic agouti-related peptide (AGRP) mRNA and neuropeptide Y (NPY) mRNA. It has been hypothesized that this profile of hypothalamic gene expression contributes to the obese phenotype caused by leptin deficiency. To assess if reversal of obese phenotype by adrenalectomy entails normalization of hypothalamic gene expression, male wild-type and ob/ob mice were adrenalectomized (with saline supplementation) or sham adrenalectomized at 2 months of age. Mice were sacrificed 2 weeks after adrenalectomy, during which time food intake and body weight were monitored daily. After sacrifice, hypothalamic gene expression was assessed by Northern blot analysis as well as in situ hybridization. In wild-type mice, adrenalectomy significantly decreased AGRP mRNA but did not significantly influence POMC or NPY mRNA. In ob/ob mice, adrenalectomy reduced the levels of plasma glucose, serum insulin and corticosterone, and food intake toward or below wild-type levels, and it restored hypothalamic POMC and AGRP mRNA but not NPY mRNA to wild-type levels. These studies suggest that adrenalectomy reverses or attenuates the obese phenotype in ob/ob mice, in part by restoring hypothalamic melanocortin tone toward wild-type levels. These studies also demonstrate that factors other than leptin may play a major role in regulating hypothalamic melanocortin function.

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin.

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased > 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.

Roles of second-messenger systems and neuronal activity in the regulation of lordosis by neurotransmitters, neuropeptides, and estrogen: a review.

Many neurotransmitters and neuropeptides can affect the rodent feminine sexual behavior, lordosis, when administered in the ventromedial hypothalamus (VMH), midbrain central gray (MCG), or other brain regions. A survey of the electrophysiological and biochemical actions of these neural agents revealed that there is a very consistent association between lordosis facilitation with both the activation of the phosphoinositide (PI) pathway and the excitation of VMH and MCG neurons. In contrast, lordosis inhibition is associated, less consistently, with alterations of the adenylate cyclase (AC) system and the inhibition of neuronal activity. The findings that lordosis could be facilitated by going beyond membrane receptors and directly activating the PI pathway, suggest that this second-messenger pathway is a common mediator for the lordosis-facilitating agents. Furthermore, as in the case of stimulating membrane receptors, direct activation of this common mediator also requires estrogen priming for lordosis facilitation. Therefore, it is likely that the PI pathway is modulated by estrogen in the permissive action of estrogen priming. Indeed, a literature review shows that estrogen can affect selective isozymes of key enzyme families of the PI pathway at various levels. Such selective modulations, at several levels, could easily alter the course of a PI cascade; thence, the eventual functional outcome. These findings prompt us to propose that estrogen enables lordosis to be facilitated by a selective modulation of the PI pathway.

Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting.

Agouti-related protein (AGRP) is a homologue of the agouti gene product and, when overexpressed, promotes obesity. Like neuropeptide Y (NPY) messenger RNA (mRNA), AGRP mRNA is produced in the hypothalamic arcuate nucleus and is elevated in leptin-deficient ob/ob and leptin-resistant db/db mice. These data suggest that AGRP mRNA might be affected by leptin and nutritional status in parallel with NPY mRNA. To test this hypothesis, we examined if AGRP mRNA would be, like NPY mRNA, inhibited by leptin injections and stimulated by fasting. AGRP mRNA was elevated in ob/ob mice about 5-fold compared with wild-type controls and was significantly inhibited by leptin treatment, as assessed by Northern blot analysis. In wild-type mice, AGRP mRNA was increased at least 13-fold by a 2-day fast, as assessed both by Northern blot analysis and in situ hybridization. In ad lib fed db/db mice, AGRP mRNA was elevated about 8-fold compared with ad lib fed wild-type controls, and was further increased by fasting in db/db mice. These data suggest that AGRP mRNA and NPY mRNA respond similarly to leptin and fasting.

Estrogen receptor messenger RNA expression in rat hypothalamus as a function of genetic sex and estrogen dose.

Previously, we showed that estrogen receptor (ER) messenger RNA (mRNA) levels are decreased in cells of the mediobasal hypothalamus of ovariectomized (OVX) female rats following an acute estradiol treatment. Here, we examined whether the level of ER mRNA remains depressed in the continued long-term presence of estradiol, and questioned if there is a systematic relationship between the concentration of estradiol and the decrease in ER mRNA level. OVX female rats were implanted for 2 weeks with silastic capsules containing various concentrations of estradiol. Tissue sections were hybridized with a [3H] single-stranded DNA probe prepared from the region of the rat ER complementary DNA corresponding to the steroid binding domain, and relative mRNA level was assessed by counting grains over cells in specific hypothalamic nuclei. Estradiol induced a dose-dependent decrease in ER mRNA levels. Message levels declined in the ventrolateral aspect of the ventromedial nucleus (VLVM) by 57% and in the arcuate nucleus by 62% at the highest hormone concentrations used. Thus, ER mRNA down-regulation in female rat hypothalamus exhibits orderly dose dependence at a time following hormone treatment which ensures the system is at steady state. A second study determined if there exist differences in basal levels of ER mRNA expression between castrated (CAS) females and males, and if estradiol can down-regulate ER mRNA levels in male hypothalamus. CAS rats of both sexes were exposed acutely to estradiol benzoate (EB) for different periods of time. Again, in females, EB significantly decreased ER mRNA levels in VLVM by 55% (18 h) and in the arcuate nucleus by 74% (18 h). Interestingly, control CAS males had significantly lower basal ER mRNA levels than OVX females (52% lower than female levels in VLVM; 56% in arcuate), suggesting a sex difference in constitutive expression levels. Moreover, EB failed to down-regulate significantly ER message levels in males. There was no significant effect of sex or EB treatment on ER mRNA levels in medial amygdala. Thus, the second study shows sex differences and brain-region specificity in hormonal regulation of ER mRNA. These findings show that differences in basal levels and regulation of ER mRNA could be a substrate for sex differences in ER concentrations in the hypothalamus of the rat, and further raise the possibility of sex differences in concentrations of nuclear proteins related to the control of ER gene expression.

Age-correlated and ovary-dependent changes in relationships between plasma estradiol and luteinizing hormone, prolactin, and growth hormone in female C57BL/6J mice.

Impairments in the estradiol (E2)-induced surge of LH occur as aging female rodents lose reproductive cycles. These and other impairments can be substantially attenuated by ovariectomy of the young adult. In female rats, plasma PRL tends to increase with age, whereas in male rats, GH tends to decrease; PRL is regulated by E2, whereas GH is probably not. To determine if age-correlated neuroendocrine impairments are accompanied by altered neuroendocrine sensitivity to E2, the relationships among plasma E2, LH, PRL, and GH were assessed in 6- and 12-month-old cycling and 18-month-old acyclic female C57BL/6J mice given E2 implants. An additional 18-month-old group, ovariectomized at 6 months of age, was examined to determine if age-correlated changes in sensitivity to E2 are ovary dependent. One week after ovariectomy, mice were given different sized E2 implants which generated a physiological range of plasma E2. Three weeks after implantation, plasma E2 correlated positively with implant size, uterine weight, and PRL, but correlated negatively with LH in each age group; age did not affect plasma E2 levels. The suppression of LH by E2 decreased progressively with age. Conversely, the elevation of PRL by E2 increased with age. These effects of age were largely prevented by ovariectomy at 6 months of age. Plasma GH decreased slightly with age, but was not significantly affected by E2; old mice ovariectomized when young had lower GH levels than previously intact old mice. GH also correlated positively with LH in all age groups. We conclude that neuroendocrine responses to E2 are altered with age even before estrous cycles are lost. Sensitivity to E2 may either increase or decrease, depending on the function. These effects of age can be attenuated by prolonged ovariectomy. Thus, chronic exposure to ovarian E2 during normal reproductive cycles may alter neuroendocrine sensitivity to E2, which may lead to age-correlated impairments in reproductive function.

Hyperphagia and weight gain after gold-thioglucose: relation to hypothalamic neuropeptide Y and proopiomelanocortin.

Genetic obesity is associated with increased neuropeptide Y (NPY) messenger RNA (mRNA) and decreased POMC mRNA in the hypothalamus of ob/ob and db/db mice, or impaired sensitivity to alphaMSH (derived from POMC) in the yellow agouti mouse. Acquired obesity can be produced by chemically lesioning the hypothalamus with either monosodium glutamate (MSG) in neonates or gold thioglucose (GTG) in adult mice. The present study examined whether elevated NPY mRNA and/or decreased POMC mRNA in the hypothalamus are associated with obesity due to hypothalamic lesions. GTG injection into adult mice produced a profound obese phenotype, including hyperphagia, increased body weight, and increased leptin mRNA and peptide, in association with reduced hypothalamic NPY mRNA and POMC mRNA. MSG treatment produced virtual elimination of NPY mRNA in the arcuate nucleus and a reduction of hypothalamic POMC mRNA, and led to elevated leptin. MSG pretreatment did not attenuate GTG-induced hyperphagia and obese phenotype. These results do not support a role for NPY-synthesizing neurons in the arcuate nucleus in mediating hypothalamic acquired obesity, but are consistent with the hypothesis that decreased activity of hypothalamic neurons synthesizing POMC play a role in mediating hypothalamic obesity.

Two-dimensional gel autoradiographic analysis of the acute effects of estradiol on protein synthesis in the female rat ventromedial nucleus in vivo.

Estradiol (E2) alters protein synthetic events in the ventromedial nucleus (VMN) of the hypothalamus to promote lordotic behavior in the female rat. This study analyzed the acute changes induced by E2 in proteins synthesized in the VMN in vivo as measured by 35S incorporation into protein, two-dimensional gel electrophoresis, autoradiography, and computerized optical densitometry. Ovariectomized rats received vehicle or E2, Hamilton syringes were placed stereotaxically in the VMN bilaterally, and 0.9 mCi 35S-labeled methionine and cysteine was infused over 1 h. After 6 h, rats were killed, and VMN samples were subjected to isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resultant gels underwent autoradiography, and the optical density of each of 240 spots was quantitated using a flat-bed laser scanner. Using a quantitation algorithm based on the linearity and reproducibility of the scanner and film, 123 spots were analyzed for changes in optical density and relative mol wt (MW) and isoelectric point (pI) induced by E2. The spot induced most prominently by E2 had a MW of 70K and a pI of 6.0, confirming previous results. Three spots were quantitatively induced by E2: 1) a 47K MW, pI 5.4 phosphoprotein (96% induction); 2) a 45K MW, pI 5.2 protein (72% induction); and 3) a 100K MW, pI 5.5 protein (82% induction). Two spots increased pI with E2: 1) a 110K MW protein increased from pI 5.4 to 5.5; and 2) a 50K MW protein increased from pI 6.3 to 6.4. Under these conditions and using a quantitative algorithm, only a small number of proteins synthesized in the VMN were induced by E2 in vivo. E2 also exerts effects on the posttranslational modification of another select group of proteins. These proteins may subserve at least part of the physiological effect of E2 in the VMN.

Pituitary and hypothalamic glucose-6-phosphate dehydrogenase: effects of estradiol and age in C57BL/6J mice.

Activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH) were measured in the mediobasal hypothalamus and pituitary gland of C57BL/6J mice throughout their lifespan. Activities of pituitary G6PDH, pituitary 6-PGDH, and hypothalamic G6PDH increase with age in female mice, assayed 7 days after ovariectomy, but not in intact females or males. Pituitary G6PDH specific activity is increased by middle-age (13-14 months) in females, before the onset of acyclicity, and remains elevated throughout the acyclic phases of persistent vaginal cornification and persistent diestrus. This increase in activity is ovary dependent, because it can be prevented by long term (12-month) ovariectomy. The increased activity is not linked to pituitary tumorigenesis and does not result from trapped blood cells, as evaluated by studies with 51Cr-labeled erythrocytes. Responses to physiological levels of estradiol (E2) were analyzed with a graded series of chronic polyethylene implants or after a single sc injection in ovariectomized mice aged 5-22 months. The responsiveness of pituitary G6PDH to E2 is not altered during aging. Young cycling (6 months old) and older acyclic mice (19 months old) displaying persistent vaginal cornification show equivalent increases of about 100% in G6PDH specific activity after chronic E2 treatment and similar time courses of induction after a single E2 injection. Pituitary G6PDH is maximally induced (30% increase) by 48 h after E2 injection in all age groups. In addition, the rates of decline in pituitary G6PDH specific activity after ovariectomy are similar in young and older mice (half-life, 4 days). The specific activity of G6PDH in the mediobasal hypothalamus and blood is unaffected by E2 administration. The relatively low doses of E2 used here fail to alter 6-PGDH specific activity in pituitary or brain. These findings indicate that female reproductive senescence in mice is not associated with generalized losses of sensitivity and responsivity to E2 throughout the neuroendocrine axis.

Reproductive senescence in female C57BL/6J mice: ovarian impairments and neuroendocrine impairments that are partially reversible and delayable by ovariectomy.

Ovarian and neuroendocrine impairments were examined before and after the age-correlated loss of estrous cycles in C57BL/6J female mice. The role of ovarian secretions in inducing neuroendocrine impairments before and after the loss of estrous cycles was also examined by determining neuroendocrine impairments after prolonged ovariectomy. Young (6-month-old) cycling, middle-aged (12-, 14-, and 16-month-old) cycling or acyclic, and old (18-month-old) acyclic mice were used. Ovarian impairments were assessed by grafting old ovaries into young hosts. Neuroendocrine impairments were assessed by grafting young ovaries into middle-aged and old hosts, ovariectomized either at grafting or 2 months before, and by inducing a LH surge in mice that had been ovariectomized for 4 days, 1 month, or 2 months. One group of 16-month-old and one group of 18-month-old mice were also ovariectomized at 6 months of age; these groups were used to examine the steroid-induced LH surge. The LH surge was induced by inserting sc a single priming estradiol (E2) implant, followed by two more (surge-inducing) implants 6 days later; 33 h after the second implantation, mice were decapitated (1800 h). The number of estrous cycles supported by middle-aged and old ovaries grafted into young hosts was reduced by more than 90% compared with that in young ovaries. The number of estrous cycles supported by middle-aged and old hosts given young ovarian grafts was reduced by 60% and 90%, respectively, compared with that in young hosts. Middle-aged and old hosts also had progressively longer estrous cycles than young hosts. Levels of the E2-induced LH surge in middle-aged and old mice were also reduced by 60% and 90%, respectively, compared with levels in young mice. In old acyclic mice, ovariectomy for 2 months partially reversed impairments in the LH surge and partially restored the ability to support cycles with young grafts; these functions, normally 90% impaired in old mice, were only 60% impaired after prolonged ovariectomy. Moreover, ovariectomy 2 months before grafting in old hosts resulted in shorter estrous cycles and 60% fewer pituitary adenomas. In middle-aged cycling mice, ovariectomy for 2 months did not affect impairments in the LH surge or in the ability to support estrous cycles with young grafts; these functions remained 60% impaired in middle-aged mice ovariectomized for 2 months. If mice were ovariectomized when young, the age-correlated impairments of the E2-induced LH surge at 16 months were largely prevented.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen increases HIP-70/PLC-alpha messenger ribonucleic acid in the rat uterus and hypothalamus.

In the ventromedial hypothalamus (VMH) of female rats, estrogen induces a protein isoform, HIP-70, whose sequence is identical to a protein reported to be phosphoinositol-specific phospholipase-C-alpha (PLC-alpha). Since previous studies explored induction only at the protein level, we examined both the distribution of HIP-70/PLC-alpha mRNA in various tissues and the effects of estrogen on HIP-70/PLC-alpha mRNA. Using slot blot analysis, we found that HIP-70/PLC-alpha mRNA is most abundant in pituitary, uterus, and VMH of female rats compared with other brain regions and tissues. Since these are target tissues for estrogen action, we examined the effects of estrogen on the abundance of HIP-70/PLC-alpha mRNA in these areas. Levels of HIP-70/PLC-alpha mRNA increased greater than 3-fold in the uterus 18 h after estrogen treatment. HIP-70/PLC-alpha mRNA in the VMH also increased about 35% 3 h after estrogen treatment. In situ hybridization corroborated the induction in the ventrolateral ventromedial hypothalamus. No effect of estrogen was observed on pituitary PLC-alpha mRNA. These results indicate that estrogen does increase HIP-70/PLC-alpha mRNA levels in certain tissues. Since the induction of HIP-70/PLC-alpha mRNA in VMH is relatively modest compared to the much larger induction of the HIP-70 protein isoform, regulation of HIP-70/PLC-alpha may entail both pre- and posttranslational mechanisms. Because members of the PLC family catalyze the hydrolysis of phosphatidyl inositol, potentially activating several secondary mediators (intracellular Ca2+, protein kinase-C, and eicanosoids), this second messenger pathway may mediate some effects of estrogen.

Fasting regulates hypothalamic neuropeptide Y, agouti-related peptide, and proopiomelanocortin in diabetic mice independent of changes in leptin or insulin.

Fasting increases hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AGRP) messenger RNA (mRNA) and reduces hypothalamic POMC mRNA, and is also characterized by a reduction in plasma leptin, insulin, and glucose, each of which has been implicated in the regulation of hypothalamic gene expression. To further evaluate the roles of leptin, insulin, and glucose in mediating effects of fasting, we examined hypothalamic gene expression in nondiabetic and streptozotocin (STZ)-induced diabetic mice both under ad lib fed and 48-h fasted conditions. In both diabetic and nondiabetic mice, fasting stimulated hypothalamic NPY and AGRP mRNA and inhibited hypothalamic POMC mRNA and adipose leptin mRNA. However, in diabetic mice fasting had no effect on plasma leptin and insulin while decreasing plasma glucose, whereas in nondiabetic mice fasting decreased plasma leptin, insulin, and glucose. Furthermore, in nondiabetic fasted mice, NPY and AGRP mRNA were higher, and POMC mRNA and plasma glucose were lower, than in diabetic ad lib fed mice, even though insulin and leptin were similar in these two groups. These data are consistent with the hypothesis that although leptin and insulin regulate hypothalamic gene expression, glucose or other factors may have independent effects on hypothalamic and adipose gene expression under conditions of low insulin and leptin.

Differential effects of estradiol and 16 alpha-hydroxyestrone on pituitary and preoptic estrogen receptor regulation.

16 alpha-Hydroxyestrone (16OHE1), an endogenous metabolite of estradiol (E2), binds to the estrogen receptor (ER) with low affinity, but is estrogenic in various bioassay systems. 16OHE1 binds covalently to the ER in vitro, exhibits prolonged estrogenic bioactivity in vivo, and has been implicated in several estrogen-dependent diseases. This study examined the effects of 13 days of continuous infusion of E2 or 16OHE1 on lordotic behavior, pituitary growth, and ER regulation in the cytosolic and nuclear fractions of the pituitary and preoptic area of both sexes. Finally, simultaneous pituitary nuclear exchange assays and enzyme immunoassays were performed to search for covalent 16OHE1-ER complexes in vivo. E2 induced lordosis and pituitary growth in both sexes, while 16OHE1 was only slightly less effective. While E2 treatment increased nuclear ER concentrations 2-fold vs. control values, it decreased both cytosolic and total (cytosolic plus nuclear) ER concentrations in pituitary and preoptic area by approximately 3-fold vs. control values in both sexes by exchange assay. In contrast, 16OHE1 did not decrease total pituitary ER concentrations and only minimally decreased total preoptic ER concentrations. Simultaneous exchange assay and immunoassay of pituitary nuclear extracts demonstrated proportionate increases in ER levels in female vs. male and in E2-treated vs. 16OHE1-treated rats. The ratios of (ER enzyme immunoassay divided by ER-exchange) for each rat were similar regardless of metabolite administration. The correlation of individual measurements implied that ER localized to the nuclear fraction by either E2 or 16OHE1 retained both exchangeability and immunoassayability to similar extents, but did not support the presence of 16OHE1-ER covalent complexes. The results of this study suggest that 16OHE1 has significant estrogenic bioactivity, as manifest by its effects on lordosis and pituitary growth, but, in contrast to E2, does not decrease pituitary ER concentrations and only minimally decreases preoptic ER concentrations. This property may be important in the proposed pathogenetic action of 16OHE1 in estrogen-dependent disease.

The regulation of luteinizing hormone and prolactin in C57BL/6J mice: effects of estradiol implant size, duration of ovariectomy, and aging.

The induction of a LH surge by estradiol (E2) implants was characterized in ovariectomized C57BL/6J mice. Various times after ovariectomy mice were given a priming E2 implant, followed by an LH surge-inducing E2 implant, and were sampled 30 h later at darkness. The magnitude of the E2-induced LH surge was influenced by the postovariectomy interval, sizes of the implants, and age. Mice ovariectomized for 30-60 days before E2 implantation displayed larger surges than those ovariectomized for 4 days. Priming implants yielding 10 pg E2/ml plasma permitted the subsequent induction of vigorous LH surges, whereas no LH surges were observed with slightly larger priming implants that yielded 15 pg E2/ml. The size of the surge-inducing implant was correlated with the size of the subsequent LH surge. However, regardless of implant size, aging mice (8 vs. 13 months old) had smaller LH surges. Sequential daily LH surges were not observed under any conditions, suggesting that mice differ from rats and hamsters in their regulation of LH by E2. Plasma PRL was slightly elevated in the afternoon just before the LH surge, but returned to basal levels during the LH surge, indicating an uncoupling of the LH and PRL surges. The two-stage E2 implantation protocol for inducing LH surges by physiological levels of E2 allows more detailed examination of the priming vs. surge-inducing effects of E2.

Evidence that glucose metabolism regulates leptin secretion from cultured rat adipocytes.

Circulating leptin secreted from adipocytes is correlated with fat mass and plasma insulin concentrations in humans and rodents. Plasma leptin, insulin, and glucose decrease during fasting and increase after refeeding; however, the underlying mechanisms regulating the changes of leptin secretion are not known. To investigate the role of insulin-stimulated glucose metabolism in the regulation of leptin secretion, we examined the effects of insulin and inhibitors of glucose transport and metabolism on leptin secretion from rat adipocytes in primary culture. Insulin (0.16-16 nM) increased leptin secretion over 96 h; however, the increase in leptin was more closely related to the amount of glucose taken up by the adipocytes (r = 0.64; P < 0.0001) than to the insulin concentration per se (r = 0.20; P < 0.28), suggesting a role for glucose transport and/or metabolism in regulating leptin secretion. 2-Deoxy-D-glucose (2-DG), a competitive inhibitor of glucose transport and phosphorylation, caused a concentration-dependent (2-50 mg/dl) inhibition of leptin release in the presence of 1.6 nM insulin. The inhibitory effect of 2-DG was reversed by high concentrations of glucose. Two other inhibitors of glucose transport, phloretin (0.05-0.25 mM) and cytochalasin-B (0.5-50 microM), also inhibited leptin secretion. Inhibition of leptin secretion by these agents was proportional to the inhibition of glucose uptake (r = 0.60 to 0.86; all P < 0.01). Two inhibitors of glycolysis, iodoacetate (0.005-1.0 mM) and sodium fluoride (0.1-5 mM), produced concentration-dependent inhibition of leptin secretion in the presence of 1.6 nM insulin. In addition, both 2-DG and sodium fluoride markedly decreased the leptin (ob) messenger RNA content of cultured adipocytes, but did not affect 18S ribosomal RNA content. We conclude that glucose transport and metabolism are important factors in the regulation of leptin expression and secretion and that the effect of insulin to increase adipocyte glucose utilization is likely to contribute to insulin-stimulated leptin secretion. Thus, in vivo, decreased adipose glucose metabolism may be one mechanism by which fasting decreases circulating leptin, whereas increased adipose glucose metabolism would increase leptin after refeeding.