Nancy LaPaglia

Peripubertal paternal EtOH exposure

Fetal alcohol syndrome usually implies effects on the offspring of maternal EtOH consumption during gestation, with fewer reports addressing the impact of paternal exposure on the progeny. One previous report has dealt with the impact of EtOH exposure on peripubertal male rats as a model of teenage drinking and the deleterious effects on the offspring. We report here findings examining the effect of 2 mo of EtOH feeding on male animals as they progressed through puberty on their ability to impregnate EtOH-naive female rats and characteristics of the subsequent litters. The EtOH-imbibing fathers weighted significantly less than pairfed controls and animals ingesting a non-EtOH liquid diet ad libitum. Nevertheless, they were able to mate successfully, although fecundity was significantly reduced. The number of successful pregnancies, defined as carried to term, was diminished from 92% in controls to 75% in EtOH-fed animals (p<0.05). There was increased paternal testicular oxidative injury demonstrated by enhanced lipid peroxidation, protein oxidation, and decreased ratio of reduced to oxidized glutathione. The litter size of the EtOH-exposed males was reduced by 46%. The average litter size was 12.4±1.5 pups/litter in ad libitum animals, virtually identical to the 12.5±0.6 pups/litter in the pair fed controls. This is in sharp contrast to the 6.7±0.1 pups/litter from the paternal EtOH matings (p<0.001). There was an increase in the average individual weight of pup offspring of paternally EtOH-exposed animals (p<0.01 vs pair-fed controls and p<0.05 vs ad libitum). Curiously, the male-to-female pup ratio was altered with a higher preponderance of male offspring from EtOH-fed fathers. There were no gross malformations noted among the pups. Insulin-like growth factor-1 levels in the pups at 10 d of age were unaltered between the groups. However, leptin was significantly elevated in the EtOH offspring. It appears that chronic EtOH exposure in the peripubertal fathers subsequently decreases fecundity and that this may be mediated by testicular oxidative injury, perhaps leading to accelerated germ cell apoptosis.

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 α‐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, alendronates 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.

The response of the hypothalamic-pituitary-gonadal axis to fasting is modulated by leptin.

Reproductive function is intimately related to caloric consumption. During fasting states, the hormones regulating reproduction, those of the hypothalamic‐pituitary–gonadal axis, in particular, are severely altered. With the exciting observations that the obese (ob) gene product, leptin, may also modulate neuroendocrine functions, we examined leptins ability to prevent the consequences of fasting on reproductive hormones. Two groups of male rats, aged 65 days old, were either fasted and saline‐injected or fasted and leptin‐treated for approximately three days. Another group was given free access to rat chow. Leptin was able to prevent the fasting‐induced fall of serum testosterone. Similar to testosterones dependence on leptin, leptin concentrations were somewhat dependent on testosterone. Castration accelerated the normal, age‐related increase in serum leptin. Leptin also prevented the fasting‐induced fall in luteinizing hormone (LH). The increase of β‐LH mRNA seen in the fasting state was prevented by leptin. There were no differences noted in luteinizing hormone releasing hormone (LHRH) mRNA among any of the groups. While neither fasting nor fasting plus leptin caused changes in serum prolactin, the increase in prolactin mRNA seen in fasted animals was prevented by leptin treatment. These data support the hypothesis that leptin plays a specific role in mediating the response of reproductive hormones to the nutritional status of the organism.

EtOH disrupts female mammalian puberty: age and opiate dependence.

The major drug of abuse among teenagers in the United States continues to be ethanol (EtOH), but use is seen in children as young as nine. In the studies reported here, the impact of EtOH on biologic and hormonal parameters of puberty was assessed in female rats. Rats were fed a liquid diet containing EtOH, pair fed an identical liquid diet containing dextrimaltose instead of EtOH, or fed a liquid diet not containing EtOH ad libitum. Feeding was started at 21, 25, or 28 d of age. EtOH markedly delayed the age at vaginal opening (34.5±0.5 d in controls vs 48.5±2.4 d in EtOH animals; p<0.001), delayed the age at first estrous (40.9±0.6 d in controls vs 61.2±2.6 d in EtOH animals; p<0.001), increased the length of the estrous cycle, and decreased the number of proestrous days. EtOH, concomitant with reduced ovarian and uterine weight, decreased serum estradiol and progesterone. Associated with these changes in ovarian hormones there was a selective increase in follicle-stimulating hormone, but not luteinizing hormone. EtOH consistently reduced insulin-like growth factor-1. In general, EtOH-induced disruption was more severe the younger the animals were at the start of feeding. Opiate receptor blockade with naltrexone completely prevented the EtOH-induced delay in vaginal opening. The impact of EtOH on female puberty is dramatic, is an emerging public health problem, and deserves more study.

Impact of acute and chronic ethanol exposure on prolactin in both male and female rats

The deleterious effects of ethanol (EtOH) on reproduction have been well documented. This disruption is usually associated with alterations in prolactin (PRL) levels, which is relevant since this hormone is an important participant in the reproductive system. Reported EtOH-induced changes in PRL (i.e., stimulation or inhibition) have varied. These differences may have been owing to the gender or age/sexual maturity of the animal and the mode of the administration of EtOH. Therefore, to clarify the impact of EtOH on PRL, a series of experiments were conducted utilizing rats of both genders, exposed to EtOH acutely or chronically, as adults and as they progressed through puberty. In general, in younger animals of both genders, EtOH depressed serum PRL whether given acutely or chronically. In adult males, acute EtOH actually stimulated PRL levels while chronic administration had no effect. In adult females, EtOH’s effect was highly dependent on the stage of the estrous cycle in which EtOH was given and during which PRL was measured. In conclusion, our studies have shown that the PRL response to EtOH is dependent on the gender and age/sexual maturity of the animals as well as on the mode of administration.

Effects of Chronic Ethanol (EtOH) Administration on Pro-Inflammatory Cytokines of the Hypothalamic-Pituitary-Gonadal (HPG) Axis in Female Rats

We and others have investigated the effects of acute and chronic ethanol (EtOH) administration on function of the hypothalamic-pituitary-gonadal (HPG) axis in female rats, consistently finding EtOH to be detrimental. There are now substantial data that pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6), have anti-reproductive effects. If EtOH increased levels of these cytokines, such data would be consistent with, though not necessarily prove, a cytokine mediated mechanism for EtOH’s deleterious effects on reproduction. Young adult female Sprague Dawley rats were used. In the experiment reported here, the Lieber DeCarli diet was used, with animals fed a 36% EtOH containing diet or pair fed an identical diet which contained dextrimaltose instead of EtOH. This was done for 4 to 6 weeks. TNFα and IL-6 were measured in the hypothalamus, pituitary, and ovary by ELISA. EtOH exposure resulted in significant increases in TNFα and IL-6 in hypothalami, pituitaries, and ovaries. The data reported here are the first to show consistent stimulatory effects of EtOH exposure on cytokines in the reproductive axis of female rats. Because the effects of these cytokines are generally anti-reproductive, these data provide a rational for more rigorous testing of the notion that part of EtOH’s deleterious HPG effects may be due to such immuno-endocrine interactions.

The effect of acute ethanol (EtOH) exposure on protein kinase C (PKC) activity in anterior pituitary

Alterations in the protein kinase C (PKC) pathway may interrupt anterior pituitary luteinizing hormone (LH) synthesis and/or secretion, which may impair normal reproductive function. Work by our laboratory and others has shown that EtOH has profound deleterious effects on the regulation of the hypothalamic-pituitary-gonadal (HPG) axis. The present study focuses on PKC translocation from the cytosol to the membrane of anterior pituitary after acute EtOH exposure. Serum levels of LH were measured at three time points (15, 30, and 90 min) after an IP injection of either saline or 3 g/kg EtOH in adult castrated male rats. LH levels dropped significantly (p < 0.03) in EtOH-injected compared to saline-injected control animals. In the same animals, EtOH significantly suppressed PKC localization at its active site at the pituitary cell membrane (p < 0.05). These findings suggest that the mechanism of EtOHs suppression of LH is mediated, at least in part, through a decrease in PKC translocation to the anterior pituitary cell membrane.

Effects of insulin on hepatic inflammation induced by ethanol and burn injury in a murine model of critical illness

In recent, landmark clinical trials, insulin to maintain euglycemia in critically ill patients improved clinical outcomes, including decreased all-cause mortality. Novel antiinflammatory effects of insulin have recently been described. Thermal injury is an excellent model of critical illness. The addition of ethanol to the model is of great clinical relevance because nearly 50% of the patients admitted to hospitals for burn injuries have ethanol in their circulation. Utilizing a murine model of critical illness (ethanol and skin burn), we tested the hypothesis that insulin treatment in ethanol-exposed, burn-injured mice reduced hepatic inflammation, a potential mechanism for the benefit of insulin. Adult male C57BL/6 mice were given a single intraperitoneal injection of ethanol or saline, were given a 15% total body full-thickness skin burn, or were sham-burned and killed 24 hours later. In each group, half the animals were given subcutaneous injections of the long-lasting basal insulin glargine; the other half, the appropriate vehicle. Hepatic inflammatory markers, including polymorphonuclear infiltration, a chemokine, an important adhesion molecule, proinflammatory cytokines, and nuclear factor &kgr;B, were measured, and all were increased by ethanol and/or burn. These increases were prevented by insulin. An antiinflammatory cytokine was reduced by ethanol and/or burn. Insulin prevented this decrease. Thus, insulin has a substantial antiinflammatory effect, and this may underlie its dramatic clinical benefit in critical illness.

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.

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 dold 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.