Ethanol-induced hypogonadism is not dependent on activation of the hypothalamic-pituitary-adrenal axis.

The well-characterized suppression of the male reproductive unit after ethanol (EtOH) exposure has been speculated to be partially due to activation of the hypothalamic-pituitary-adrenal (HPA) axis. The subsequent corticosterone elevation could result in hypogonadism via suppression of hypothalamic LHRH, pituitary LH, or a direct gonadal effect. To directly examine this possibility, adult male Sprague Dawley rats were either adrenalectomized (ADX) or sham ADX. The ADX animals were given low dose corticosterone via replacement pellet, resulting in a steady level of serum corticosterone. The sham ADX (adrenal intact) animals were implanted with placebo pellets. Half of both groups were then exposed to EtOH by I.P. injection on two consecutive days to mimic an acute binge-drinking model. The other half was given saline I.P., serving as controls. In the adrenal intact animals, EtOH caused the expected rise in corticosterone, and fall in luteinizing hormone (LH) and testosterone. In the ADX animals, where constant levels of corticosterone were maintained by pellet implantation, EtOH resulted in similar LH and testosterone reduction. These results suggest that suppression of the reproductive axis is independent of the activation of the HPA unit.

Alendronate administration and skeletal response during chronic alcohol intake in the adolescent male rat.

Alendronate is an aminobisphosphonate that inhibits bone resorption in osteoporotic humans and rats but does not induce osteomalacia. Several bisphosphonates, including alendronate, also have direct positive actions on osteoblasts, bone formation, and mineralization. We studied the effects of alendronate on skeletal development in adolescent male rats during chronic alcohol intake. Four groups of age- and weight-matched male Sprague-Dawley rats (35 days of age) were fed the Lieber-DeCarli diet containing 36% of calories as EtOH (E), the EtOH diet plus 60 mg/kg alendronate (EA) every other day intraperitoneally (ip), an isocaloric diet (I), or the isocaloric diet plus 60 mg/kg alendronate (IA) every other day ip. Body weight, femur length, serum levels of osteocalcin (OC), insulin-like growth factor 1 (IGF-1), testosterone, and luteinizing hormone (LH); femur distal metaphyseal and middiaphyseal bone mineral density (BMD) and tibial metaphyseal gene expression for alpha-1-type I collagen (Col I), OC, and bone alkaline phosphatase (AP); and femur strength by four-point bending to failure were measured after 28 days of feeding and alendronate injections. Serum alcohol levels at death were 156 +/- 13 mg/dl (E) and 203 +/- 40 mg/dl (EA). Alendronate given to alcohol-fed rats increased metaphyseal BMD by more than 3-fold over rats fed alcohol alone. Alendronate given to isocaloric pair-fed rats increased metaphyseal BMD by more than 2.5-fold over rats fed the isocaloric diet alone. Cortical BMD was reduced by alcohol but was increased by alendronate. Alcohol consumption reduced serum IGF-1 levels, and alendronate increased IGF-1 levels in alcohol-fed rats. Serum OC, testosterone, and LH were unaffected by alcohol and alendronate. Quantitative dot blot hybridization using rat complementary DNA (cDNA) probes and normalization against 18S subunit ribosomal RNA (rRNA) levels revealed no changes in tibial metaphyseal gene expression for type I collagen, osteocalcin, or alkaline phosphatase. Alcohol significantly reduced the biomechanical properties of the femurs that were partially compensated by alendronate. Chronic alcohol consumption uncouples formation from ongoing resorption, and resorption is inhibited by alendronate. However, alendronate's positive effects on osteoblast-mediated mineralization during chronic alcohol consumption point to the potential use of bisphosphonates in the treatment of decreased bone formation secondary to alcohol-induced diminished osteoblast function.

Impact and reversibility of chronic ethanol feeding on the reproductive axis in the peripubertal male rat.

Teenage drinking continues to be a major problem in the United States as well as abroad. A significant depression in serum testosterone in adolescents who consume EtOH has been well described. In the male rodent model, a similar fall in testosterone has been reported, and prevention with the opiate blocker naltrexone has been demonstrated. To explore further the impact of chronic EtOH exposure on the reproductive axis in peripubertal rats, we designed this study specifically to define whether or not there was recovery after abstinence by examining reproductive hormones and their genes during and after EtOH exposure. Peripubertal male rats 35 d old were fed an EtOH-containing diet or a calorically matched control diet for 60 d. A third group was fed the control liquid diet ab libitum. EtOH was then withdrawn and all animals were fed standard rat chow and water ad libitum for an additional 3 mo. The EtOH-imbibing animals were found consistently to weigh less than their pair-fed mates and liquid diet ad libitum animals. Serum testosterone levels and testicular weights were significantly decreased by EtOH whereas serum estradiol levels were higher, suggesting enhanced peripheral conversion by EtOH. Spermatogenesis, assessed by histological parameters, was unaltered by EtOH. Serum luteinizing hormone levels were not different among the groups. Hypothalamic luteinizing hormone-releasing hormone mRNA levels were unaffected by EtOH. During the 3-mo recovery period, all the changes reversed, with a significant increase noted in testosterone. All other parameters remained the same among the groups. Thus, although chronic EtOH exposure in the peripubertal age period results in significant reproductive alterations, there is complete recovery on withdrawal.

The role of gonadectomy and testosterone replacement on thymic luteinizing hormone-releasing hormone production.

We and others have identified luteinizing hormone-releasing hormone (LHRH) in cells of the immune system in both animals and humans. LHRH is an immunostimulant, and testosterone is an immunosuppressant. Because testosterone is known to modulate the concentrations of hypothalamic LHRH, we wondered whether testosterone might also alter the concentrations of rat thymic LHRH. Two weeks after castration or sham castration, adult male rats were implanted with either vehicle or testosterone capsules. All animals were killed 4 days after capsule implantation. Thymic LHRH concentration increased significantly in castrated animals. Testosterone replacement prevented this increase. The concentration of the LHRH precursor, proLHRH, decreased significantly, but testosterone replacement prevented this decrease. Steady-state concentrations of LHRH mRNA were not changed by castration or by hormonal replacement. In contrast to the post-castration increase in thymic LHRH, LHRH content of the hypothalamus decreased significantly. Whereas concentrations of LHRH were lower in the thymus than in the hypothalamus, proLHRH concentrations were much greater in the thymus. These data suggest that gonadal manipulation modulates LHRH molecular processing and its tissue concentration in the thymus in addition to those in the hypothalamus, and that the regulation of LHRH molecular processing by testosterone in the hypothalamus is different from that in the thymus.

Reversal of ethanol-induced testosterone suppression in peripubertal male rats by opiate blockade.

Teenage drinking is a major problem in the United States, as well as abroad. Besides psychosocial implications, ethanol (EtOH) has detrimental effects on the reproductive system. Clinical problems associated with reduced reproductive hormones include osteoporosis, decreased muscle function, anemia, altered immune function, prostate involution, and decreased reproductive abilities. Education coupled with strategies aimed at preventing these deleterious consequences even in the face of continued EtOH intake is extremely important. We have tested the possibility that naltrexone, a drug currently used in patients to decrease alcohol craving, might also prevent the fall in the male hormone, testosterone, caused by EtOH exposure. Rats aged 35 days old (prepubertal), 45 days old (midpubertal), and 55 days old (late pubertal) were injected (intraperitoneally) with either saline, EtOH, naltrexone, or EtOH plus naltrexone. In the two older age groups, EtOH significantly suppressed testosterone, which was prevented by administration of naltrexone. In the youngest animals, there was no treatment effect presumably due to low basal levels of testosterone. EtOH similarly reduced luteinizing hormone (LH), but this suppression was not prevented by naltrexone. There was no consistent effect of any treatment on hypothalamic concentration of pro-LH releasing hormone (RH) (LHRH), LHRH, or on steady-state levels of LHRH mRNA. We conclude that, as animals progress through puberty, EtOH suppresses LH and testosterone. The testosterone decline can be prevented by opiate blockade with naltrexone, an effect primarily seen at gonadal level. Thus, naltrexone, a drug already used clinically to reduce EtOH intake, also has protective physiological effects on the endocrine system.

Leptin alters the response of the growth hormone releasing factor- growth hormone--insulin-like growth factor-I axis to fasting.

Proper nutritional status is critical for maintaining growth and metabolic function, playing an intimate role in neuroendocrine regulation. Leptin, the recently identified product of the obese gene, may very well be an integral signal which regulates neuroendocrine responses in times of food deprivation. The present study examines leptin's ability to regulate hormonal synthesis and secretion within the GRF-GH-IGF axis in the adult male rat during almost 3 days of fasting. Serum levels of GH and IGF-I were drastically suppressed by fasting. Daily leptin administration was able to fully prevent the fasting-induced fall in serum GH. Leptin failed to restore IGF-I to control levels, however, suggesting possible GH resistance. Fasting caused an insignificant increase in GH mRNA, while leptin injections significantly increased steady-state levels of this message. The GRF receptor (GRFr) message was not altered with fasting or leptin treatment. Leptin also exhibited effects at the hypothalamic level. Fasting induced a sharp fall in GRF mRNA expression and leptin injections partially prevented this fall. However, there were no observed changes in the hypothalamic GRF content. These results provide evidence that leptin may function as a neuromodulator of the GRF-GH-IGF axis communicating to this hormonal system the nutritional status of the animal.

Castration differentially regulates nitric oxide synthase in the hypothalamus and pituitary.

Mammalian reproductive function is under control of the integrated hypothalamic-pituitary-gonadal (HPG) axis. Castration in male rats has been utilized as an effective tool to investigate hormonal interactions in the mammalian HPG axis. Recently, nitric oxide (NO) has been suggested to play a role in HPG hormonal regulation. In order to gain further insight into the function of the NO-NOS system in reproductive neuroendocrine control, particularly in the gonadal feedback regulation of the hypothalamic-pituitary unit, we examined steady state levels of nNOS mRNA, nNOS protein, and the important physiological index, NOS enzyme activity, of the intrinsic NOergic system in both hypothalamus and pituitary in castrated male rats and their sham-operated counterparts one week after surgery. In the pituitary, we found a significant four-fold increase in nNOS mRNA, p < 0.0003 compared to sham. Castration also resulted in a four-fold rise in pituitary nNOS protein, p < 0.02 compared to sham. Pituitary NOS enzyme activity was stimulated 2 fold, p < 0.003 after castration. In the hypothalamus, conversely, we observed no significant castration-modulated difference in either nNOS mRNA, nNOS protein or NOS enzyme activity. Thus, it appears that the hypothalamic NO-NOS system is either not required for hypothalamic adaptations to castration, although important in the release of LHRH under normal physiological conditions, or alternatively, the hypothalamus may become more sensitive to the effects of NO in the castrated state. In the pituitary, NO may attenuate the gonadotropin response to castration as a local balancing mediator.

Effect of chronic ethanol on reproductive and growth hormones in the peripubertal male rat.

Ethanol (EtOH) has previously been shown to have profound effects on various endocrine systems. The present study further investigates the action of EtOH on testosterone and on the GH-IGF-I axis. Since these hormones are particularly important in male rats progressing through puberty, we examined the effect of 10 days of EtOH treatment at three different ages (35, 50 and 65 days old) as male rats progressed through puberty into adulthood. After 10 days of feeding a 6% EtOH liquid diet, serum testosterone levels were markedly decreased in all three ages (P < 0.02 at 35 days, P < 0.01 at 50 days and P < 0.03 at 65 days). IGF-I was assessed and was differentially affected at each age. At 35 days IGF-I levels were suppressed by EtOH (P < 0.0002), at 50 days no change was apparent, and at 65 days levels were significantly higher in EtOH-treated (P < 0.01) compared with liquid-fed controls. The levels of IGF-I in the EtOH-treated animals paralleled pituitary GH mRNA levels with a significant fall in the expression of GH mRNA levels noted at 35 days (P < 0.04), no change at 50 days and a significant rise observed at 65 days (P < 0.03). At the hypothalamic level, GH-releasing hormone (GRF) mRNA was significantly reduced in the two younger EtOH-treated age groups compared with controls (P < 0.04 at 35 days; P < 0.02 at 50 days). At 65 days of age, EtOH did not alter GRF mRNA levels. No EtOH-induced changes were seen in GRF content at any age. These observations indicate definite age-related alterations in hormonal gene expression and circulating serum hormone levels and emphasize the importance of studying these critical peripubertal ages after chronic EtOH exposure.

Ethanol, growth hormone and testosterone in peripubertal rats.

The deleterious effects of ethanol on the hypothalamic pituitary growth hormone axis in adult male humans and animals have been well documented. It is also well established that ethanol has toxic effects on testicular function in adult humans and animals. Much less is known, however, about the effects of ethanol on the growth hormone (GH) axis and testicular function in adolescence. Recent studies have established that adolescent problem drinking is a widespread and growing threat to the health of young people in the United States. In the present study, therefore, we investigated if acute ethanol exposure in peripubertal male Sprague-Dawley rats altered normal pituitary and testicular function. Serum levels of GH and testosterone were measured at 1.5, 3, 6, and 24 h after a single i.p. injection of either saline or 3 g/kg body weight ethanol. Histologic analysis as well as serum testosterone levels allowed us to assign animals to either early puberty (35-day-old animals), mid-puberty (41-day-old animals), or young adult (51- and 66-day-old animals) status. Ethanol produced significant decrements in serum testosterone in the 51- and 66-day-old animals, with a trend toward suppression in the 41-day-old group. Furthermore acute ethanol administration significantly decreased serum GH (P < 0.0001 by 3 way ANOVA) demonstrating a significant effect of ethanol on serum GH in all age groups and at all time points studied when compared with saline injected controls (P < 0.01 by Turkey's studentized range test). Despite this significant fall in peripheral GH levels, there was no decrease in either GH mRNA or growth hormone-releasing factor (GRF) mRNA levels nor in hypothalamic concentration of GRF peptide. We conclude that, as in adult animals, acute exposure to ethanol causes a prolonged and severe decrement in serum GH which is possibly mediated at the level of secretion. In addition, there is attenuation in testosterone secretion. These data are all the more important since GH and testosterone play critical roles in organ maturation during this stage of development.