Vinod K. Srivastava

Effects of lead (Pb) exposure during gestation and lactation on female pubertal development in the rat.

Lead (Pb) can delay sexual maturation; however, the mechanism and critical time of insult are not clearly defined. Therefore, we assessed maternal Pb levels during low-level gestational and/or lactational exposure, as well as blood and tissue Pb in developing fetuses in relation to the subsequent detrimental effects of Pb on puberty-related hormones and the onset of female puberty. Adult Fisher 344 female rats were gavaged daily with either a 1-ml solution of PbAc containing 12 mg/ml Pb or an equal volume of sodium acetate (NaCl), for the controls, from 30 days prior to breeding until their pups were weaned at 21 days. By cross-fostering at the time of birth, the pups were either exposed to PbAc or NaAc during gestation only, lactation only, or during both gestation and lactation. Pb delayed the timing of puberty and this delay was associated with suppressed serum levels of insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), and estradiol (E(2)). Liver IGF-1 mRNA was not affected, suggesting that Pb altered translation and/or secretion of IGF-1. We reported previously that peripherally derived IGF-1 acts at the hypothalamic level to facilitate LH release at puberty; hence, we suggest that the action of Pb in decreasing circulating IGF-1 contributes to the delayed puberty. The detrimental effects occurred regardless of the developmental time of exposure, although gestational exposure appeared more sensitive to the effects of Pb. Also, the effects noted were with blood Pb levels less than previously reported and these levels are relevant to human health concerns.

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.

Lipopolysaccharide-induced leptin release is neurally controlled

Our hypothesis is that leptin release is controlled neurohormonally. Conscious, male rats bearing indwelling, external, jugular catheters were injected with the test drug or 0.9% NaCl (saline), and blood samples were drawn thereafter to measure plasma leptin. Anesthesia decreased plasma leptin concentrations within 10 min to a minimum at 120 min, followed by a rebound at 360 min. Administration (i.v.) of lipopolysaccharide (LPS) increased plasma leptin to almost twice baseline by 120 min, and it remained on a plateau for 360 min, accompanied by increased adipocyte leptin mRNA. Anesthesia largely blunted the LPS-induced leptin release at 120 min. Isoproterenol (β-adrenergic agonist) failed to alter plasma leptin but reduced LPS-induced leptin release significantly. Propranolol (β-receptor antagonist) produced a significant increase in plasma leptin but had no effect on the response to LPS. Phentolamine (α-adrenergic receptor blocker) not only increased plasma leptin (P < 0.001), but also augmented the LPS-induced increase (P < 0.001). α-Bromoergocryptine (dopaminergic-2 receptor agonist) decreased plasma leptin (P < 0.01) and blunted the LPS-induced rise in plasma leptin release (P < 0.001). We conclude that leptin is at least in part controlled neurally because anesthesia decreased plasma leptin and blocked its response to LPS. The findings that phentolamine and propranolol increased plasma leptin concentrations suggest that leptin release is inhibited by the sympathetic nervous system mediated principally by α-adrenergic receptors because phentolamine, but not propranolol, augmented the response to LPS. Because α-bromoergocryptine decreased basal and LPS-induced leptin release, dopaminergic neurons may inhibit basal and LPS-induced leptin release by suppression of release of prolactin from the adenohypophysis.

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.

Ethanol blocks the central action of IGF-1 to induce luteinizing hormone secretion in the prepubertal female rat.

Insulin-like growth factor-1 (IGF-1) is emerging as a signal of peripheral origin capable of acting centrally to induce luteinizing hormone (LH) secretion and accelerate the initiation of female puberty. Since we have shown previously that ethanol (ETOH) can suppress prepubertal LH release and delay female puberty, we hypothesized that these detrimental effects might be due, at least in part, to the drugs ability to alter the central actions of IGF-1. Thus, we have investigated the effects of ETOH on IGF-1 induced LH release in vivo, and on IGF-1 induced prostaglandin-E2 (PGE2) and LH-releasing hormone (LHRH) release in vitro from rats during the juvenile phase of development as well as from rats during the early stage of first proestrus. For the in vivo experiment three initial jugular blood samples were taken at 10-min. intervals from all rats, then the animals received either a 3g/Kg dose of ETOH or an equal volume of saline by gastric gavage. The rats were subsequently left undisturbed for 90 min. to allow time for ETOH absorption, then a single blood sample was drawn from each rat. Finally, IGF-1 (200 ng/3 microl) was microinjected into the third ventricle of all animals and five more blood samples were withdrawn at 10 min. intervals. We demonstrated that IGF-1 induced the release of LH (p<0.01) in the saline controls from rats in both phases of pubertal development. Conversely, this effect of IGF-1 was blocked by ETOH in both developmental groups. For the in vitro experiment, median eminences (MEs) were dissected, then incubated in the presence or absence of ETOH (50 mM). The amount of PGE2 and LHRH released was measured from the same samples following the addition of IGF-1 (100 ng/ml). As above, similar responses were observed from rats in both developmental phases. IGF-1 stimulated the release of PGE2 (p<0.001) and LHRH (p<0.01) from the incubated nerve terminals in the absence of ETOH; however, these effects were blocked by the presence of ETOH. Thus, these combined in vivo and in vitro results suggest that ETOH can acutely block IGF-1 induced LH release during the juvenile-peripubertal transition period, and that this is a centrally mediated action which is due to the diminished formation of PGE2 resulting in decreased LHRH release.

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: Effects of caloric restriction

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.

Manganese stimulates luteinizing hormone releasing hormone secretion in prepubertal female rats: hypothalamic site and mechanism of action

We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone‐releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30‐day‐old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N‐monomethyl‐l‐arginine (NMMA); 300 or 500 μm) or a soluble guanylyl cyclase (sGC) inhibitor (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ); 100 or 250 μm) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 μm) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 μm MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300–500 μm NMMA to the medium did not block Mn2+‐stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 μm MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+‐stimulated secretion of LHRH in a dose‐dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 μm) inhibited Mn2+‐induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.

Influence of estradiol on insulin-like growth factor-1-induced luteinizing hormone secretion.

Several studies suggest an interrelationship between estradiol (E2) and insulin-like growth factor-1 (IGF-1) at the hypothalamic level. The present study was designed to discern if the capability of IGF-1 to release LH and influence the timing of female puberty is influenced by E2. Twenty-eight-day-old female rats were ovariectomized (OVEX), then implanted with a third ventricular (3V) cannula. Two weeks later, these animals received subcutaneous (s.c.) injection of oil, or either one or two injections of E2 in the form of estradiol benzoate (1 microg). Forty-eight hours later, four basal blood samples were drawn then the animals received IGF-1 (200 ng) or saline via the 3V and four more blood samples were taken. Results indicated that E2 replacement lowered basal LH levels and IGF-1 induced a significant LH release in only animals that had E2 levels above 20 pg/ml. These levels of E2 were also associated with increases (p<0.05) in the expression of both IGF-1 receptor (IGF-1R) mRNA and protein. In order to further support the hypothesis that the action of IGF-1 at the time of puberty is influenced by E2, 24-day-old intact female rats received s.c. injection of sesame oil or 0.1 microg of E2. The next day, the E2-treated animals also received twice daily s.c. injections of either IGF-1 (500 ng) or saline until vaginal opening (VO) occurred. The animals that received E2 plus IGF-1 showed VO at 31.1 days, which was 2.5 days earlier (p<0.01) than E2-treated animals and 4 days earlier (p<0.001) than IGF-1-treated and saline control animals. Taken together, these results indicate that the hypothalamic action of IGF-1 to stimulate LH release and advance female pubertal development is dependent upon the influence of E2.

Short-term alcohol administration alters KiSS-1 gene expression in the reproductive hypothalamus of prepubertal female rats.

BACKGROUND Kisspeptins bind to the G-protein-coupled receptor (GPR54) to activate hypothalamic luteinizing hormone releasing hormone (LHRH) secretion at the time of puberty. Alcohol (ALC) causes depressed prepubertal LHRH release, resulting in depressed luteinizing hormone (LH) secretion and delayed puberty. Because KiSS-1 and GPR54 are important to the onset of puberty, we assessed the effects of chronic ALC administration on basal expression of these puberty-related genes within the reproductive hypothalamus, as well as hormones and transduction signaling pathways contributing to their activity. METHODS Immature female rats were fed a liquid diet containing ALC for 6 days beginning when 27 days old. Controls received either companion isocaloric liquid diet or rat chow and water. Animals were decapitated on day 33, in the late juvenile stage of development. Blood was collected for the assessment of serum hormone levels. Brain tissues containing the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei were obtained for assessing expression of specific puberty-related genes and proteins. RESULTS KiSS-1 mRNA levels in the AVPV and ARC nuclei were suppressed (p < 0.001) in the ALC-treated rats. GPR54 gene and protein expressions were both modestly increased (p < 0.05) in AVPV nucleus, but not in ARC nucleus. Alcohol exposure also resulted in suppressed serum levels of insulin-like growth factor-1 (IGF-1), LH, and estradiol (E(2)). As IGF-1, in the presence of E(2), can induce expression of the KiSS-1 gene, we assessed the potential for ALC to alter IGF-1 signaling in the reproductive hypothalamus. IGF-1 receptor gene and protein expressions were not altered. However, protein expression of phosphorylated Akt, a transduction signal used by IGF-1, was suppressed in the AVPV (p < 0.05) and ARC (p < 0.01) nuclei. CONCLUSIONS Alcohol causes suppressed KiSS-1 gene expression in the reproductive hypothalamus; hence, contributing to this drugs ability to cause suppressed LHRH secretion and disruption of the pubertal process. We suggest that this action, at least in part, is through altered IGF-1 signaling.

Lipopolysaccharide-Induced Leptin Synthesis and Release Are Differentially Controlled by Alpha-Melanocyte-Stimulating Hormone

Objective: Since α-melanocyte-stimulating hormone (α-MSH) inhibits the synthesis and release of proinflammatory cytokines and stimulates the synthesis and release of anti-inflammatory cytokines, and leptin is a cytokine that has anti-inflammatory actions in the presence of lipopolysaccharide (LPS), we hypothesized that α-MSH increases leptin synthesis and release. Methods: α-MSH or 0.9% NaCl (saline) were injected intraperitoneally 15 min prior to intravenous injection of 0.5 ml of saline or LPS (0.15 mg/kg). Thereafter, repeated blood samples were withdrawn over a period of 6 h and plasma leptin concentrations determined. The rats were sacrificed at 6 h and leptin mRNA was measured in epididymal fat pads. Results: Plasma leptin concentrations of the saline-injected control group were unaltered during the 6 h, whereas in the LPS group, leptin was unaltered between 0 and 30 min and thereafter progressively increased between 30 and 360 min by 2.5-fold. α-MSH slightly increased plasma leptin concentrations by 15 min and then increased them further by 120 min, after which they declined towards baseline. The pattern of plasma leptin concentrations in the α-MSH + LPS group was similar to that of the LPS group, except that higher concentrations were observed at 120 min in the rats injected with α-MSH + LPS. LPS increased leptin mRNA by 3-fold at 6 h, whereas it was unaffected in the MSH-treated animals. On the contrary, α-MSH completely blocked the LPS-induced leptin mRNA. Conclusions: Our results suggest that α-MSH increased leptin release without altering its synthesis, but when LPS increased release and synthesis of leptin, α-MSH, although further increasing release, blocked the enhanced synthesis of leptin elicited by LPS.