Insulin-like growth factor-1 stimulation of hypothalamic KiSS-1 gene expression is mediated by Akt: effect of alcohol.

Kisspeptin, as well as insulin-like growth factor-1 (IGF-1), act centrally to stimulate luteinizing hormone-releasing hormone (LHRH) secretion at puberty. IGF-1 can induce KiSS-1 gene expression as an early pubertal event; however, the signaling pathway mediating this effect is not known. Since alcohol (ALC) blocks IGF-1 induced LHRH release acutely, we assessed whether this drug could affect IGF-1 stimulated prepubertal KiSS-1 gene expression following a binge type of exposure. Immature female rats were administered either ALC (3 g/kg) or water via gastric gavage at 07.30 h. At 09.00 h the ALC and control groups were subdivided where half received either saline or IGF-1 (200 ng) into the third ventricle. A second dose of ALC (1.5, 2 and 3 g/kg) or water was administered at 11.30 h. These regimens produced moderate blood alcohol concentrations of 77, 89 and 117 mg/dl, respectively, over the time course of the experiment. Rats were sacrificed 6 h after the IGF-1 injection and tissues containing the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei were collected. IGF-1 stimulated (P<0.01) KiSS-1 gene expression in the AVPV nucleus at 6 h, but did not affect expression of the kisspeptin receptor, GPR54. While ALC did not alter basal expression of either gene, its dose dependently blocked IGF-1-induced KiSS-1 gene expression in the AVPV nucleus. No changes were observed in the ARC nucleus. Assessment of IGF-1 signaling indicated that the acute administration of IGF-1, ALC, or both did not alter the basal expression of IGF-1 receptor protein. However, IGF-1 stimulated (P<0.05) phosphorylated Akt protein over basal levels, an action blocked by ALC. Our results indicate that the IGF-1 induction of KiSS-1 gene expression is mediated by Akt activation, and that ALC alters this important prepubertal action of IGF-1.

Insulin-like growth factor-I activates KiSS-1 gene expression in the brain of the prepubertal female rat.

KiSS-1 gene expression has been shown to increase as puberty approaches, and its peptide products, kisspeptins, are involved in LHRH secretion at puberty. Factors contributing to increased KiSS-1 expression, however, have not been identified; thus, the purpose of this study was to assess whether IGF-I could induce transcription of this gene in prepubertal female rats. IGF-I or saline was centrally administered to immature rats that were killed 2, 4, and 6 h later. Real-time PCR revealed that IGF-I induced (P < 0.01) KiSS-1 gene expression at 6 h in a tissue fragment that contained both the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. Subsequently, the AVPV and ARC nuclei were separated to assess whether region-specific effects could be identified. IGF-I stimulated (P < 0.01) KiSS-1 gene expression in the AVPV nucleus at 6 h after injection, with no change observed in the ARC nucleus. Serum estradiol (E2) levels were not altered at any time point after IGF-I, demonstrating that the increased KiSS-1 expression observed was not caused by an elevation in E2. Additionally, the IGF-I action to induce KiSS-1 gene expression in the AVPV nucleus was further demonstrated when the IGF-I was administered systemically. E2 appears to play an important permissive role because 1-d ovariectomized rats responded to IGF-I with increased (P < 0.01) KiSS-1 expression, whereas, 20 d after ovariectomy, when the E2 levels had fallen below assay sensitivity, the IGF-I was unable to induce KiSS-1 expression. The IGF-I effect was further demonstrated by showing that the IGF-I receptor antagonist, JB-1, blocked the IGF-I-induced increase in KiSS-1 expression. Collectively, these data indicate that IGF-I is an activator of the KiSS-1 gene in the prepubertal female rat.

Low level lead (Pb) exposure during gestation and lactation: assessment of effects on pubertal development in Fisher 344 and Sprague-Dawley female rats.

Studies using both Fisher 344 and Sprague-Dawley (SD) rat lines have shown that gestational and/or lactational maternal lead (Pb) exposure causes delayed reproductive maturation in their respective female offspring. Because these studies utilized different experimental regimens for dosing and for monitoring Pb levels, it has not been possible to determine which rat line provides the best model for low level Pb toxicity studies. This study was designed to address this issue. Adult Fisher and SD female rats were dosed with either a solution of PbAc containing 12 mg of Pb/ml or sodium acetate (NaAc) for controls. Dosing began 30 days prior to breeding and continued until their pups were weaned at 21 days of age. At the time of breeding and through weaning the blood lead (BPb) levels in the Fisher dams averaged 37.3 microg/dl and the SD dams averaged 29.9 microg/dl. Pb delayed the timing of puberty (p < 0.01) in Fisher offspring, and suppressed serum levels of luteinizing hormone (LH, p < 0.001) and estradiol (E2, p < 0.01). These effects did not occur in the SD offspring. Doubling the dose given to the SD rats increased their BPb levels to 62.6 microg/dl, yet there were still no effects noted. These results indicate that Fisher offspring are more sensitive to maternal Pb exposure with regard to puberty related insults than are SD rats, suggesting that the Fisher line may be a more reliable rodent model to study the effects of low level Pb toxicity.

Chronic effects of prepubertal ethanol administration on steroidogenic acute regulatory protein in the rat ovary.

BACKGROUND: A mitochondrial protein, steroidogenic acute regulatory protein (StAR), plays an essential role in steroidogenesis by facilitating delivery of cholesterol across the mitochondrial membrane. Because ethanol (EtOH) causes suppressed estradiol (E2) secretion in prepubertal female rats and rhesus monkeys, we evaluated the effects of chronic EtOH administration on prepubertal ovarian StAR. METHODS: Rats were implanted with a gastric cannula on day 24 and began receiving control or EtOH diets on day 28. At 0800 hr on day 33, the experimental groups were subdivided. Half of the EtOH-treated animals received a subcutaneous injection of pregnant mare serum gonadotropin (PMSG; 15 IU), and the other half received an injection of saline. The chow-fed and liquid-diet control groups were also subdivided, with half receiving the PMSG and the other half receiving saline. Eight hours after the respective injections, the animals were killed, and their ovaries and blood were collected. RESULTS: The ovaries from EtOH-treated rats showed decreased basal expression of both the 3.8-kb (p < 0.05) and 1.7-kb (p < 0.01) StAR transcripts. PMSG-stimulated animals not exposed to EtOH showed a more than 2-fold increase (p < 0.01) in the ovarian levels of both transcripts. Western blot analysis revealed that EtOH exposure decreased (p < 0.001) the basal expression of StAR protein, which paralleled the decrease in basal StAR messenger RNA. PMSG induced an increase (p < 0.001) in the levels of StAR protein, and this effect was blunted (p < 0.01) by EtOH. These changes observed in ovarian StAR protein were paralleled by decreases in serum pregnenolone and E2. CONCLUSIONS: These results demonstrate for the first time that ovarian StAR is a target for the chronic action of EtOH to alter prepubertal steroidogenesis, resulting in suppressed serum E2 secretion during a critical time of development.

Acute effects of ethanol on steroidogenic acute regulatory protein (StAR) in the prepubertal rat ovary.

BACKGROUND: Steroidogenic acute regulatory protein (StAR) is a 30 kDa mitochondrial protein that plays an essential role in steroid hormone biosynthesis by facilitating delivery of cholesterol across the mitochondrial membrane, where side chain cleavage occurs to initiate ovarian steroidogenesis. Because ethanol (EtOH) suppresses estradiol secretion in prepubertal female rats, we evaluated the effects of EtOH on prepubertal ovarian StAR. METHODS: At 0700 hr, 28-day-old female rats were gavaged with saline or a 3 g/kg dose of EtOH. At 0800 hr, half of each of these two groups was treated with 15 IU of pregnant mare serum gonadotropin (PMSG). At 1000 hr, a 2 g/kg dose was administered to maintain moderately elevated blood alcohol levels. At 1600 hr, all of the animals were killed by decapitation, and blood and ovaries were collected for measurement of serum pregnenolone and estradiol and for ovarian StAR gene and protein expression. RESULTS: Northern blot analysis showed two major transcripts of 3.8 and 1.7 kb of ovarian StAR mRNA. The ovaries from EtOH-treated rats showed decreased (p < 0.01) basal expression of both 3.8 and 1.7 kb StAR transcripts. PMSG-stimulated animals showed a more than 4-fold increase (p < 0.001) in the levels of both transcripts, when compared with ovaries from animals that received saline or EtOH only. Conversely, in EtOH-treated animals, the PMSG-stimulated expression of the 1.7 kb transcript was blocked, and the increase in the 3.8 kb StAR transcript was blunted (p < 0.05 vs. PMSG). Western blot analysis revealed that EtOH exposure also depressed (p < 0.01) the basal expression of StAR protein. PMSG-stimulated animals showed an increase (p < 0.001) in levels of StAR protein, and this was blocked (p < 0.01) by EtOH. These changes observed in ovarian StAR mRNA and protein were paralleled by changes in serum pregnenolone and estradiol. Specifically, acute EtOH exposure suppressed (p < 0.05) the basal levels of both steroids. Furthermore, PMSG-stimulated animals showed an increase in the production of pregnenolone (p < 0.05) as well as estradiol (p < 0.01), and EtOH blocked this stimulatory action of PMSG on both steroids. CONCLUSION: These results demonstrate for the first time that EtOH is capable of altering ovarian StAR expression, which contributes to the detrimental effect this drug has on ovarian steroidogenesis during prepubertal development.

Effects of ethanol on leptin secretion and the leptin-induced luteinizing hormone (LH) release from late juvenile female rats.

BACKGROUND: Chronic ethanol (EtOH) exposure lowers serum insulin-like growth factor-1 (IGF-1) and luteinizing hormone (LH) levels and also delays female puberty, similar to the deficits in the reproductive system that occur during leptin deficiency. Leptin administration restores fertility and gonadotropin secretion in the ob/ob mouse and can induce recovery of reproductive function in food-restricted animals. This study assessed the effects of EtOH on serum leptin levels, and whether exogenous leptin administration could restore IGF-1 and LH levels in the EtOH-treated animals. METHODS: In the first study, 29-day-old female rats were divided into control and EtOH-treated groups, each of which received their respective diet regimen for 5 consecutive days. The EtOH-treated animals were subdivided and received an intraperitoneal injection of either leptin (100 microg/0.1 ml) or saline twice daily. Control animals also received intraperitoneal saline injections twice daily. On day 34, animals were killed, and serum leptin, LH, and IGF-1 were measured by RIA. In a second study we assessed the acute effects of a single 3 g/kg dose of EtOH on the ability of leptin to act centrally to induce LH release. For this, leptin (1 microg) was administered via a third ventricular (3V) cannula and blood sampling via jugular cannula. In a third experiment, animals were again subjected to a chronic feeding regimen. When 34 days old, they were killed and the anterior pituitaries removed and incubated in a static incubation system for 60 min to establish basal LH release, then for an additional 60 min in medium containing leptin (10(-7) M). RESULTS: Chronic EtOH exposure lowered serum leptin (p < 0.01), IGF-1 (p < 0.01), and LH (p < 0.05) levels. Leptin administration to EtOH-treated animals did not restore serum IGF-1 levels. This peptide did, however, effectively restore LH levels to normal, but did not advance the timing of puberty. Acute EtOH administration was found to block leptin-induced LH release following central administration of the peptide. Conversely, anterior pituitaries from control and 5-day EtOH-treated animals that were incubated in vitro released (p < 0.01) equal amounts of LH in response to leptin (10(-7) M). CONCLUSIONS: These data demonstrate that EtOH administration not only can suppress peripheral levels of leptin, but also blocks its central action to facilitate LH secretion. Although replacement of leptin can reverse the EtOH-induced suppression of LH by a direct action at the level of the pituitary, it cannot elevate serum IGF-1; a peripheral signal that acts centrally to stimulate LH releasing-hormone (LHRH)/LH release during the juvenile-peripubertal transition period, and thus accelerates the initiation of female puberty. These results demonstrate further the complex actions and interactions of multiple hormones involved in the pubertal process and the vulnerability of their actions to the toxic effects of EtOH.

Alcohol's effects on female puberty: the role of insulin-like growth factor 1.

Research suggests that alcohol consumption during early adolescence may delay the onset of female puberty. Alcohol's effect on sexual development is associated with altered function of insulin-like growth factor 1 (IGF-1). This hormone, which is produced in the liver, travels through the bloodstream to the brain, where it helps coordinate overall physical growth with the maturation of the reproductive system. Long-term alcohol consumption inhibits the production of IGF-1 in the liver. Short-term alcohol administration alters IGF-1 function within the brain, ultimately suppressing the release of specific reproductive hormones that initiate puberty. Large proportions of young girls develop drinking habits that place them at risk for alcohol-related endocrine disorders at a crucial time in female pubertal development.