Rat adult offspring serum lipoproteins are altered by maternal consumption of a liquid diet.

Palatable liquid diets for the administration of ethanol (EtOH) to animals have proven to be a major advance for the study of the effects of EtOH consumption under conditions of isocaloric nutrition of the control animals. Using a liquid diet, the original aim of the reported studies was to examine the effect of maternal EtOH consumption during pregnancy on the lipoprotein (Lp) profiles of the adult offspring measured by means of nuclear magnetic resonance spectroscopy. However, initial data suggested that compared to a maternal chow diet, the basal maternal liquid diet (without EtOH) had a significant effect on specific serum Lp of the adult offspring. The adult offspring of mothers who had consumed a basal liquid diet without EtOH exhibited significant increases in their plasma triglycerides (TG) and cholesterol content compared to adult offspring whose mothers consumed a chow diet. Further, there were significant increases in the offspring's VLDL and low density Lp (LDL) subfractions' particle number, regardless of whether the maternal liquid diet was ad libitum-fed, pair-fed, or EtOH-containing. The increase in offspring plasma TG was due to increases in specific VLDL subfraction particle numbers and not to increased TG content per particle. Similarly, the increase in plasma cholesterol was the result of elevated level of the very small LDL particles but not to an increased amount of cholesterol per LDL particle. These findings should be further examined in light of the widespread use of liquid diets in research to administer EtOH, especially for studies of fetal alcohol syndrome.

Rat striatal adenosinergic modulation of ethanol-induced motor impairment: possible role of striatal cyclic AMP.

We have previously reported the involvement of the striatum in acute ethanol-induced motor incoordination and the striatal adenosinergic modulation of ethanol-induced motor incoordination through A1 receptor-mediated mechanism(s). The present study, a continuation of our previous work, was carried out to investigate the possible functional correlation between striatal cyclic AMP and ethanol-induced motor incoordination, and its modulation by striatal adenosine in Sprague-Dawley rats. Forskolin (0.1, 0.5 and 1.0 pmol), a known activator of adenylate cyclase, significantly attenuated ethanol-induced motor incoordination in a dose-dependent manner following its direct intrastriatal microinfusion. Forskolin also antagonized the accentuating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. These results suggested that ethanol-induced motor incoordination might be functionally correlated to a decrease in the striatal cyclic AMP levels and that the striatal adenosine A1 receptors might modulate ethanol-induced motor incoordination through cyclic AMP signaling mechanism(s). Further support to this hypothesis was obtained by the actual measurement of the striatal cyclic AMP levels in the same experimental conditions as in motor coordination studies using high-performance liquid chromatography with fluoroscence detection. Regardless of the method (focused microwave irradiation, cervical dislocation or decapitation into a dry ice-ethanol mixture) used to kill the animals, a significant decrease in the striatal cyclic AMP levels was observed due to ethanol. Intrastriatal adenosine A1-selective agonist, N6-cyclohexyladenosine (24 ng), caused a further significant decrease in the striatal cyclic AMP levels in the ethanol- but not in the vehicle-treated animals. The further enhancement in the ethanol-induced decrease in the striatal cyclic AMP levels by intrastriatal N6-cyclohexyladenosine, therefore, functionally correlated with the observed potentiating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. The effects of intrastriatal N6-cyclohexyladenosine+ethanol and of ethanol alone on the striatal cyclic AMP levels were blocked by intrastriatal pertussis toxin (500 ng) pretreatment, indicating the involvement of pertussis toxin-sensitive G-proteins (Gi, Go) and possibly of the adenosine A1 receptor coupled to the G-proteins in the striatum. Furthermore, ethanol alone significantly decreased the basal as well as the cyclic AMP-stimulated catalytic activities of the striatal cyclic AMP protein kinase, which were further reduced by intrastriatal N6-cyclohexyladenosine. The results of the present study therefore support an involvement of a cyclic AMP signaling pathway in the striatal adenosinergic modulation of ethanol-induced motor incoordination at the post-adenosine A1 receptor level.

Insulin resistance in adult rat offspring associated with maternal dietary fat and alcohol consumption.

Maternal diet during pregnancy has been reported to alter the offspring's ability to respond to a glucose challenge. The current studies report changes in basal and insulin-stimulated, in vitro glucose uptake in red (soleus) and white (extensor digitorum longus) muscle fiber types, as well as whole body insulin responsiveness of adult rat offspring associated with their mother's dietary fat and alcohol content during pregnancy. The offspring of Harlan-derived Sprague-Dawley female rats, dosed during pregnancy with ethanol (ETOH) via a liquid diet (35% of calories as ETOH) with either 12% or 35% of calories as fat, were compared with offspring from litters whose mothers were pair-fed an isocaloric amount of the liquid diet without ETOH. Maternal access to the liquid diets was terminated on day 20 of the pregnancies (sperm plug=day 0). The offspring were surrogate fostered within 48 h of birth to mothers which had consumed commercial chow throughout their pregnancy. Following weaning at 21 days of age, the offspring consumed only commercial rat chow and they were examined over the next 14 months for changes in glucose homeostasis as a consequence of in utero exposure to maternal dietary fat and/or alcohol. The 35% maternal fat diet resulted in both in vivo and in vitro decreases in insulin sensitivity. Thus, compared with adults whose mother's diet contained 12% fat, significant, in vitro muscle and in vivo whole body insulin resistance (measured by hyperinsulinemic-euglycemic clamping) was observed in adult rats whose mothers consumed 35% of dietary calories as fat. The addition of ethanol to the maternal 35% fat diet further reduced the offspring's red muscle tissues in vitro response to insulin, but did not affect whole body insulin sensitivity. Muscle basal and insulin-stimulated receptor tyrosine kinase activity were significantly decreased (approximately -50%) by the 35% fat maternal diet but there was no compensatory increase in serum insulin or glucose levels. Based upon both in vivo and in vitro data, these studies suggested that in utero exposure to 35% fat has a sustained effect on the adult offspring's glucose uptake/insulin sensitivity and that the effect is paralleled, at least in part, by decreased insulin receptor tyrosine kinase activity. In utero ETOH exposure resulted in the loss of basal and insulin-stimulated, in vitro glucose uptake in red muscle fibers but maternal dietary ETOH had no detectable effect on either in vivo insulin sensitivity or muscle tyrosine kinase activity.

Changes in brain glucose levels and glucose transporter protein isoforms in alcohol- or nicotine-treated chick embryos.

Suppression of fetal brain growth during pregnancy as the result of maternal smoking or alcohol consumption leads to significant problems for the offspring as well as for the society who must care for these individuals. Chronic maternal intake of cigarette smoke is frequently observed in humans and studies using animal models suggest that in utero nicotine exposure is an important component of the growth suppression that results. Similarly, maternal consumption of alcohol (ethanol) has a profound, negative effect on fetal growth. The developing fetal central nervous system (CNS) is sensitive to the growth inhibitory effect of nicotine or alcohol and morphological as well as functional CNS deficits may result from fetal exposure. Using an embryonic chick model which minimizes drug-induced changes in maternal nutrition and behavior, the studies presented here indicate that nicotine or alcohol exposure during early embryonic development inhibits brain growth to a degree comparable to that seen in the rest of the organism, i.e., there was no 'brain sparing' in this model. Glucose content per milligram tissue was markedly decreased in brains of the nicotine-treated embryos but was not significantly different in the alcohol-exposed embryos. Western blots of fetal brain glucose transporter protein isoforms showed no change in the Glut 3 transporter content in the growth suppressed brains compared to vehicle-treated brains. The Glut 1 55 kilodalton (kd) isoform protein content was significantly decreased in the nicotine-treated brains but unchanged in the ethanol-treated brains, while the reverse was true for the Glut 1 45 kd isoform. Thus, the changes in the 55 kd isoform protein content were correlated with tissue glucose levels in the ethanol- and nicotine-treated embryos.

Biochemical changes, early brain growth suppression and impaired detour learning in nicotine-treated chicks.

Fetal growth suppression associated with chronic maternal intake of cigarette smoke is frequently observed in humans and studies using animal models suggest that in utero nicotine exposure is an important component of this growth suppression. The developing fetal central nervous system (CNS) is sensitive to the growth inhibitory effect of nicotine and morphological as well as functional CNS deficits may result from fetal nicotine exposure. The studies presented here show that nicotine exposure during early embryonic development ultimately inhibits the ability of 7-11 day old chicks to learn a detour task. The brain growth suppression caused by nicotine is paralleled by a failure of the early embryo brain to express the normal developmental increase in ornithine decarboxylase (ODC) activity. This biochemical change may be germane to the mechanism of nicotine-induced growth inhibition and/or nicotine-induced behavioral changes because the appropriate expression of ODC activity is essential to normal growth and differentiation in the fetal CNS. In the chick embryo, nicotine exposure alters several important signaling pathways that regulate ODC expression. For example, nicotine exposure lowers embryonic brain glucose levels and causes significant decreases in whole brain cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels and in cyclic AMP binding proteins (protein kinase-A regulatory activity). Also, in cultured chick cells, nicotine inhibits the ability of a potent mitogen (insulin) to induce ODC activity, but, paradoxically, in ovo nicotine exposure increased insulin binding and stimulated insulin receptor autophosphorylation in brain membranes.

Indomethacin stimulation of lipid peroxidation and chemiluminescense in rat liver microsomes.

Peroxidation of endogenous lipid by liver microsomes, coupled with oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and measured as thiobarbituric acid reactive materials, is markedly stimulated in the presence of indomethacin [1-(p-chlorobenzyl)-5-methoxy-2-methyl-3-indole acetic acid] (0.1--1.0 mM). Concurrently, indomethacin enhances the lipolysis of membrane phospholipid containing arachidonic acid but has no effect on the rate of O2 uptake in these samples. The system generates a rapidly developed chemiliminescense (CL), the intensity and rate of development of which are related to indomethacin concentration. The microsomal CL generated in the presence of indomethacin is distinct from the previously reported CL in that the time required for maximum intensity development is a matter of seconds (20--180) rather than hours. The enhanced CL is believed to be due to an energy transfer reaction whereby a high energy species transfers energy to the indomethacin molecule, which, in turn, decays via chemiluminescense. An enhanced chemiluminescense was also observed when indomethacin was added to a lipoxidase system and superoxide generating system (axanthine oxidase). Based on inhibitor studies, the rapidly developed chemiluminescense of the microsomal system requires cytochrome P-450 in addition to NADPH and coordinated iron ions. The results indicate that the CL is related to neither hydroxyl free radical nor superoxide anion formation.

Alcohol metabolism and fetal hypoplasia in chick brain.

Chick embryos given a single dose of ethanol (1.0 g/kg) at the start of incubation (day 0) had widely differing levels of blood alcohol when sacrificed on day 7 and the blood alcohol levels were inversely correlated with whole body and brain weight. Clearance of the alcohol by the embryos was inhibited by simultaneous treatment with 4-methyl pyrazole and this treatment potentiated the brain growth inhibition due to ethanol. Treatment with indomethacin lowered blood alcohol levels on day 7 and protected against the growth inhibition. These data suggest that early chick embryos have varying amounts of alcohol dehydrogenase-like metabolic activity and that higher levels of this activity protect against alcohol-induced brain growth inhibition in this model. If similar variations in the ability to metabolize alcohol exist in human fetuses, it may represent a mechanism by which comparable maternal doses of alcohol produce widely varying fetal effects.

Embryonic ethanol exposure impairs detour learning in chicks.

Six groups of 30 fertile eggs each were injected with 200 microliters of either sterile water or a 37.5% (v/v) ethanol solution immediately before incubation or on days 7 or 14 of incubation. Regardless of day of injection, the ethanol groups had a lower proportion of chicks who completed pipping and hatching. Of the chicks surviving to pip and hatch, the group injected with ethanol immediately before incubation was delayed significantly in pipping compared to the other ethanol-injected groups and to the group injected with water immediately before incubation (p less than 0.05 in all cases). Also, compared to the chicks injected with water, the chicks in all ethanol groups required more trials to attain criterion on a detour learning task when tested 7-10 days after hatching (p less than 0.05).

Ethanol-induced insulin resistance suppresses the expression of embryonic ornithine decarboxylase activity.

In utero exposure to ethanol is associated with significant increases in fetal morbidity and mortality as well as with behavioral and learning problems that appear later in life. Growth suppression of the developing child is the most frequent physical effect of ethanol exposure and is correlated with specific molecular changes within the developing organism. The present report suggests that embryonic ethanol exposure suppresses the normal developmental increase in ornithine decarboxylase (ODC) activity. The loss of ODC activity during the early stages of development is dose-dependent and is correlated with the degree of growth suppression. Because ODC is the rate-limiting step for the synthesis of the polyamines and thus appears to be a focal enzyme for the regulation of growth, we have investigated the biochemical consequences of an ethanol-induced inhibition of ODC activity. Using intact chick embryos as well as cultured embryonic tissue, these studies indicate that ethanol-induced changes in tissue putrescine content result in growth suppression because a single dose of exogenous putrescine blocked the growth suppression. In cultured tissue, ethanol exposure inhibited the ability of a known trophic factor (insulin) to induce ODC activity. The loss of insulin-inducible decarboxylase activity as a result of ethanol exposure was specific to ODC, but ethanol per se had no effect on ODC activity in vitro. The data suggest that exposure to ethanol results in a resistance of the embryonic tissue to the action of insulin and thereby disrupts the molecular path by which this mitogenic compound induces the expression of ODC enzymatic activity.

Cyclic AMP-dependent protein kinase activity in the brains of alcohol-preferring (P) and nonpreferring (NP) rats.

In contrast to the reported response of inbred male mice [Beeker et al. (2)], young male rats with differing preferences for ethanol (P and NP lines) exhibited no significant change in brain cyclic AMP-binding activity following free-choice consumption of ethanol for 28 days. However, for the NP line, phosphorylation of the kinase regulatory subunit (RII) by basal kinase activity in the cytoplasmic fraction was significantly suppressed by free-choice ethanol consumption but the preferring (P) line showed no such changes. Thus the changes in phosphorylating activity appeared to be associated with differences in the animals' responses to ethanol exposure. The lower preference line (NP) consumed a smaller amount of ethanol (mean = 1.7 +/- 0.1 g/kg/24 hours) and showed a significant decrease in phosphorylating activity relative to vehicle-treated animals whereas the high preference line (P) showed no such change in kinase catalytic activity relative to controls, even though these animals consumed a significantly larger dose of ethanol (mean = 5.9 +/- 0.7 g/kg/24 hours). The data suggest that the P and NP lines differ as to their sensitivity to ethanol-induced changes in the phosphorylation of brain protein kinase regulatory subunit, an important parameter in the overall regulation of kinase activity.

The effect of embryonic ethanol exposure on detour learning in the chick.

To examine the effects of embryonic ethanol exposure on survivability, posthatch growth and detour learning, 4 groups of 30 fertile black sex-linked eggs were given 200 microliter injections of a solution of water and 0.0, 12.5, 37.5 or 50.0% (groups IC, L, M and H, respectively) ethanol (v/v) immediately preceding incubation. A fifth group of 33 eggs served as noninjected controls (NC). The results demonstrated that compared to group NC, a smaller percentage of groups M and H hatched and survived for behavioral testing, and that group H required more trials to reach criterion on the detour learning problem than did group IC. It was concluded that the teratogenic effects of ethanol are robust with respect to species and that the chick is an excellent model for studying some developmental and behavioral effects resulting from embryonic ethanol exposure.

Drinking of high concentrations of ethanol versus palatable fluids in alcohol-preferring (P) rats: valid animal model of alcoholism.

A genetically based animal model of alcoholism has been characterized in Wistar-derived rats in terms of their preference (P rats) or lack of preference (NP rats) for 10% ethanol over water. The present experiments were designed to determine: 1) whether a 10% solution of ethanol is the optimal concentration for differentiation of these lines; 2) what concentrations of ethanol are maximally preferred by P and NP rats; and 3) whether highly palatable fluids presented simultaneously with each rat's preferred solution of ethanol would alter the patterns of drinking by either the P or NP or both lines of rats. A three-bottle procedure was used to establish preference for ethanol in the presence of water as well as highly palatable solutions. The results showed that, when concentrations ranging from 3-30% were presented over a 12-day test interval, the mean absolute intake of ethanol of the P rats was 6.7 g/kg per day, with a maximum intake of 10.9 g/kg per day at the 25% concentration. These levels of intake were significantly higher than the 4.3 g/kg per day consumed during the presentation of the commonly used constant concentration of 10%. Similarly, the mean absolute intake of ethanol by the NP rats was also elevated significantly at concentrations of 15-30% (2.0 g/kg per day) above that consumed at the 10% concentration (0.4 g/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of ethanol on the metabolism of prostaglandins and related compounds.

Previous studies have shown that chronic consumption of ethanol by rats lowers the level of membrane-bound arachidonic acid (C20:4) and stimulates the in vitro measured rate of hepatic lipid peroxidation. These observations suggested that ethanol might thereby cause changes in the metabolic pathway leading to prostaglandins and related compounds. Initial studies demonstrated that chronic ethanol administration to male rats results in an impaired ability on the part of these animals to catabolize prostaglandins via renal prostaglandin dehydrogenase (PGDH) but no effect was observed on the synthesis of thromboxanes by blood platelets from these same animals. Experiments have now been carried out in an attempt to further assess the acute and chronic effects of ethanol on the metabolism of prostaglandins and prostacyclin. Generally, these results suggest a lack of an acute effect of ethanol and a dose dependency for the chronic effects.

Biochemical interactions of ethanol with the arachidonic acid cascade.

A rapidly increasing scientific literature now supports the possibility of an alcohol-prostaglandin interaction. This chapter reviews evidence for both direct and indirect biochemical interactions between ethanol and the metabolism of arachidonic acid and several related compounds. Much of the present data is based on pharmacological manipulation of prostaglandin (PG) levels by potent nonsteroid anti-inflammatory agents such as indomethacin. Indomethacin markedly alters the behavioral response to ethanol, particularly in the mouse model. These data suggest that PGs are involved in the behavioral response to acute ethanol exposure in the mouse. In other animal models, alcohol has been reported to alter blood platelet metabolism of arachidonic acid, to suppress the enzymatic degradation of PGs, and to alter the response of the adenyl cyclase system to several hormones including PGs of the "E" series. In humans, both the stimulation and inhibition of PG synthesis is reported to aid the treatment of various aspects of alcoholism. Further, PGs are reported to protect against alcohol-induced fatty liver, and both PGs and arachidonic acid protect the gastric mucosa against ethanol-induced lesions. Certainly the residual consequences of acute, excessive ethanol consumption are commonly treated with a prostaglandin synthesis inhibitor. The material in this chapter is an attempt to review the data and to discuss the molecular mechanism underlying these observations.