- Email: [email protected]
Ethanol intake during lactation
Alcohol 21 (2000) 201 ± 206
Ethanol intake during lactation II. Effects on pups' liver and brain metabolism L.M. Oyamaa, R.C. Coutob, G.E.C. Coutoc, A.R. DaÃmasod, C.M. Oller do Nascimentoa,* a
Departamento de Fisiologia, Universidade Federal de SaÄo Paulo - EPM, SaÄo Paulo, SP 04023 - 060, Brazil b Departamento de CieÃncias FisioloÂgicas, Universidade do Amazonas, Manaus, AM, 69007 - 000, Brazil c Departamento de Desporto e Atividades ComunitaÂrias, Universidade do Amazonas, Manaus, AM, 69007 - 000, Brazil d Departamento de EducacËaÄo FõÂsica e Motricidade Humana, Universidade Federal de SaÄo Carlos, SaÄo Carlos, SP, 13565 - 535, Brazil Received 28 July 1999; received in revised form 29 November 1999; accepted 13 December 1999
Abstract Lactating rats, with litters adjusted to 8 pups on day 1, were divided into 4 groups: control animals (C), which received water and Nuvilab chow ad libitum, and ethanol animals (E), which received 20% (E20), 10% (E10), or 5% (E5) ethanol diluted in the drinking water and Nuvilab chow ad libitum. On day 12 of life, the pups were weighed and decapitated. The intake of 10% and 20% ethanol solutions by the lactating rats decreased the pups' body weight and liver weight. The pups' liver ATP - citrate lyase activity was decreased in all ethanol groups. The pups' brain weight decreased in E20 only. Glucose metabolism and lactate production were studied in the pups' brain slices, which were incubated at 37°C in Krebs ± Henseleit buffer under carbogen in the presence of glucose (5 mM) plus 14C - glucose (0.04 mCi) with or without beta - hydroxybutyrate or insulin. Study of the incubated pups' brain slices showed that the intake of the 20% ethanol solution by the dams increased glucose consumption, oxidation, lactate production, and lipogenesis rate from glucose in all media studied, as compared with findings in the C group. In the pups' brain slices, the lactate production and lipogenesis rate from glucose were higher in E10 than in the C group. The addition of beta - hydroxybutyrate to the incubation medium caused a decrease in glucose oxidation in C, E5, and E20 and an increase in glucose consumption in E10. Ingestion of the 5% ethanol solution by dams decreased the pups' brain lipogenesis rate from glucose in all media studied. We concluded that the effects of maternal alcohol intake on the pups' development and metabolism are dose - dependent. High amounts of ethanol intake (10% or 20%) caused a great impairment in the pups' growth, as well as their liver and brain metabolism. The low dose (5%) did not affect the pups' body weight gain or their brain and liver weight, but it did alter brain glucose metabolism. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Alcohol; Lactation; Pups' growth; Liver; Brain; Rat
1. Introduction There has been extensive documentation that alcohol consumption during pregnancy has a detrimental effect on fetal growth and development and alters hepatic and cerebral intermediary metabolism, neurotransmitters, and lipid metabolism (Breese et al., 1994; Lancaster et al., 1982; Rawat, 1975). During lactation, the ingestion of a diet that obtained approximately one third of its calories from ethanol reduced * Corresponding author. Departamento de Fisiologia Ð Disciplina de Neurofisiologia e Fisiologia EndoÂcrina, Rua Botucatu, 862, 2° andar EdifõÂcio de CieÃncias BiomeÂdicas, Vila Clementino Ð SaÄo Paulo, SP 04023 - 060, Brazil. Tel.: +55 - 11 - 576 - 4527; fax: +55 - 11 - 570 - 7675. E-mail address: [email protected] (C.M. Oller do Nascimento).
milk production, altered milk composition (Vilaro et al., 1987), and retarded offsprings' body and central nervous system growth (Tavares -do - Carmo & Nascimento - Curi, 1990). It has also been demonstrated that alcohol administration to lactating rats inhibited suckling -induced prolactin release (Subramanian et al., 1991). Ingestion of ethanol during lactation resulted in the presence of ethanol and acetaldehyde in the milk (Guerri & Sanchis, 1986). Recently, we have shown that the effect of ethanol on dams' and pups' metabolism appears to be related to the dose of alcohol administered. High doses of ethanol (10% and 20%) ingested by the lactating rats promoted modifications in dams' metabolism and impaired pups' growth, while the intake of a low ethanol dose (5%) did not affect pups' growth despite changing dams' metabolism. In addition, it has been reported that pups receiving
0741-8329/00/$ ± see front matter D 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 9 9 ) 0 0 0 7 4 - 4
202
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
Table 1 Effect of ethanol intake by lactating rats on pups liver weight (g), protein content (mg / g), ATP - cit, and malic enzyme activities (mmol / min / 100 mg of protein) and on pups' body weight (g) day 12 after birth (g) C E5 E10 E20
Liver weight
Protein
0.59 0.03 0.55 0.02 0.36 0.02 0.29 0.01
133.4 6.76 142.45 4.56 139.33 7.18 145.04 4.88
(6) (6) (6)* (6)*
(9) (17) (16) (18)
Malic enzyme
ATP - cit
0.03 0.007 (9) 0.06 0.01 (12) 0.06 0.005 (13) 0.06 0.007 (13)
0.18 0.03 0.13 0.02 0.12 0.01 0.08 0.01
Body weight (7) (13)* (13)* (16)*
19.57 0.42 (10) 20.02 0.6 (10) 16.32 0.69 (10)* 10.83 0.51 (10)*
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
received 20% (E20), 10% (E10), or 5% (E5) ethanol solutions diluted in the drinking water and Nuvilab chow ad libitum. The mean of dams' ethanol intake (ml /100 g b.w.) on day 12 of lactation in E5, E10, and E20 was 1.38 0.08, 1.94 0.10, and 2.86 0.10, which resulted in 2.87 1.06, 43.45 11.50, and 100.66 25.30 blood ethanol concentration (mg / dl), respectively. On day 12 of life the pups were weighed and decapitated. The liver and the brain were removed, weighed, and stored in a deep freezer (ÿ70°C) for posterior analysis.
4.0 g/ kg/day of ethyl - alcohol did not exhibit significant microencephaly when compared with controls (Bonthius & West, 1988). Prenatal ethanol exposure decreases the content of neuronal glucose transporter mRNA, which would inhibit the ability of the fetal brain to take up glucose (Singh et al., 1992). In rats, a high degree of brain development and myelination occurs during the first 15 days after birth (Fando et al., 1988; Reinis & Golman, 1980). Findings from many studies from 1970 ± 1980, both in human infants and in the rat pup model, showed that the glucose and ketone bodies acetoacetate and D -(ÿ)- 3- hydroxybutyrate are taken up by the brain and used for energy production and as carbon sources for lipogenesis (Edmond, 1992). The aim of the present study was to evaluate the effect of different amounts of maternal ethanol ingestion (5%, 10%, or 20%) during lactation on pups' liver and brain metabolism. Glucose consumption and oxidation, lactate production, and lipid synthesis by pups' brain slices in vitro were also determined.
2.2. Tissue ATP -citrate lyase and malic enzymes activities For measurement of ATP -citrate lyase (ATP - cit) and malic enzymes activities, the liver and brain were homogenized at a proportion of 1:5, with the extraction medium appropriate for each enzyme, and activities were measured as described previously by Corrigan and Rider (1983) and Newsholme and Williams (1978), respectively. Tissue protein concentration was determined by the method used by Lowry et al. (1951).
2. Methods
2.3. Brain incubation
The Experimental Research Committee of the Federal Sao Paulo University approved all procedures involving animals.
Samples of brain ( 100 mg) were sliced for the incubation procedure. Incubations were performed at 37°C in 25 ml Erlenmeyer flasks equipped with a central well. The incubation medium consisted of 2.0 ml of Krebs ±Henseleit buffer containing 5 mM of glucose plus 0.04 mCi of 14C glucose. Eleven micromolar beta - hydroxybutyrate, an important energy supplier and a carbon source for lipogenesis in the developing brain (Edmond, 1992), or 0.5 U / ml insulinÐsince it has been previously demonstrated that chronic hyperinsulinemia could alter glucose utilization by several brain regions (Doyle et al., 1995) Ð was added to
2.1. Animals and procedures Wistar lactating rats were housed under constant conditions of lighting (12 h/ 12 h light/dark) and temperature (24 1°C) in individual cages with 8 pups each. On the first day postpartum, lactating dams were divided into four groups: control animals (C), which received water and Nuvilab chow ad libitum, and ethanol animals, which
Table 2 Effect of ethanol intake by lactating rats on pups' brain weight (g), protein content (mg / g), ATP - cit, and malic enzyme activities (mmol / min / 100 mg of protein) C E5 E10 E20
Weight
Protein
1.03 0.015 (6) 1.02 0.02 (6) 0.95 0.02 (6) 0.81 0.04 (6)*
66.87 4.83 72.58 2.40 64.85 4.70 64.54 4.39
(9) (10) (10) (10)
Malic enzyme
ATP - cit
0.60 0.04 (9) 0.53 0.01 (10) 0.62 0.006 (10) 0.58 0.04 (9)
0.47 0.05 0.35 0.03 0.53 0.07 0.39 0.03
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
(7) (10) (9) (9)
L.M. Oyama et al. / Alcohol 21 (2000) 201±206 Table 3 Effect of ethanol intake by lactating rats on pups' brain glucose consumption (mmol / min / 100 mg of tissue / h) Glucose C E5 E10 E20
25.63 1.65 26.9 1.88 25.76 1.56 33.29 2.19
(9) (6) (8) (10)*
Glucose + b - hydroxybutyrate
Glucose + insulin
22.05 1.87 23.42 1.88 29.01 2.74 30.57 1.97
23.82 1.43 25.34 1.70 32.27 2.94 36.22 1.74
(10) (9) (8)* (12)*
(11) (9) (8)* (8)*
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
the medium. The flasks were continuously shaken and flushed with carbogenium (O2CO2, 95%/5%) during the incubation period. After 1 h, incubation was stopped by the addition of 0.5 ml of 4N H2SO4 into the main well. Three tenths milliliter of NaOH (1N) was added to the central well for 14CO2 collection, during 2 h after the incubation was stopped. After that, brain slices were removed, and total lipids were extracted in 10 ml of chloroform ±methanol (2:1), the radioactivity in this extract representing the conversion of [1- 14C]- glucose into lipid. Rates of conversion of [1 - 14C] -glucose into 14CO2 and incorporation into the lipid fraction were expressed as mmol /h g of tissue. The incubation medium used for determination of brain glucose consumption and lactate production was the same as that used by Dubowski (1962) and Engel and Jones (1978), respectively. 2.4. Statistical analysis Results were expressed as mean SEM, and statistical comparison among groups was made by one- way analysis of variance. Differences among means were tested for significance by the Duncan's multiple range test, and p < 0.05 was taken as the level of significance. 3. Results As shown in Table 1, pups' body and liver weights decreased in E10 and E20 when compared with findings Table 4 Effect of ethanol intake by lactating rats on pups' brain glucose oxidation (mmol / g tissue / h) Glucose C E5 E10 E20
0.66 0.11 (11) 0.63 0.08 (6) 0.67 0.09 (9) 1.08 0.10 (13)*
Glucose + b - hydroxybutyrate 0.33 0.07 0.31 0.04 0.43 0.06 0.64 0.09
y
(12) (5)y (11) (13)*,y
Table 5 Effect of ethanol intake by lactating rats on pups' brain lipogenesis rate (14C - glucose incorporated in lipid / g tissue / h) Glucose C E5 E10 E20
(13) (5) (8) (14)*
Values is the mean SEM. The number of rats used in each determination are shown in parentheses. y Significantly different from glucose medium at p < 0.05. * Significantly different from control group at p < 0.05.
1.24 0.13 0.66 0.05 2.05 0.17 1.84 0.12
(12) (9)* (10)* (10)*
Glucose + b - hydroxybutyrate
Glucose + insulin
1.15 0.11 (13) 0.71 0.04 (10)* 1.53 0.16 (11)*,y 1.96 0.10 (8)*
1.44 0.14 0.89 0.09 1.57 0.16 1.79 0.16
(15) (12)* (12)y (7)
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05. y Significantly different from glucose medium at p < 0.05.
in the C group. However, the ingestion of different ethanol concentrations by the lactating rats did not alter protein content and malic enzyme activity in this tissue. On the other hand, the ATP -cit activity of pups' liver was decreased in all experimental groups. No changes were observed in protein content, nor in malic or ATP - cit enzyme activities, in pup's brain from all experimental groups, though brain weight was lower in E20 than in the C group (Table 2). Study of the incubated pups' brain slices showed that ingestion of the 20% ethanol solution by the lactating rats increased glucose consumption in all media studied, as compared with findings in the C group. When beta -hydroxybutyrate or insulin was added to the incubation medium, the consumption of glucose by the brain of E10 became higher than that of the C group (Table 3). Glucose oxidation increased in E20 when compared with that of the C group. Addition of beta -hydroxybutyrate to the incubation medium caused a decrease in glucose oxidation in C, E5, and E20. No alteration in this parameter was observed in E10 (Table 4). Ingestion of the 5% ethanol solution by the dams decreased pups' brain lipogenesis rate from glucose in all media studied. The 10% and 20% ethanol solutions increased this parameter in the presence or absence of beta hydroxybutyrate when compared with findings in the C group. In E10, the lipogenesis rate from glucose was higher when the medium contained only glucose, in relation to the other media (Table 5).
Table 6 Effect of ethanol intake by lactating rats on pups' brain lactate production (mmol / g of tissue / h)
Glucose + insulin 0.43 0.09 0.48 0.05 0.55 0.09 1.19 0.10
203
Glucose C E5 E10 E20
2.21 0.20 2.60 0.17 3.04 0.14 4.07 0.30
(11) (11) (10)* (6)*
Glucose + b - hydroxybutyrate
Glucose + insulin
2.58 0.29 2.77 0.13 2.80 0.12 3.65 0.12
2.20 0.21 2.35 0.16 2.72 0.16 4.26 0.22
(10) (8) (10) (7)*
(11) (10) (11) (7)*
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
204
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
Lactate production was increased in E20 in all studied media. In E10, lactate production was higher than that in the C group in the medium containing only glucose (Table 6). 4. Discussion The results obtained in this study showed that maternal consumption of ethanol during lactation resulted in serious modifications in pups' nutritional status and metabolism. These effects were related to the amount of ethanol ingested by dams. Our results show that the intake of 10% and 20% ethanol solutions by dams decreased pups' body weight and liver weight. The growth and development of pups are directly related to the quantity and quality of the milk. It has been previously shown that the decrease in milk production induced by a high ethanol intake during lactation could not be attributed only to the undernourishing effect of ethanol (Tavares do Carmo et al., 1999). This could be related to the fact that alcohol administration during lactation affects milk consumption by rat pups, by decreasing suckling -induced prolactin and oxytocin release (Funchs, 1969; Subramanian, 1995). Furthermore, ethanol neurotoxicity, which leads to dehydration and hyperosmolarity due to supraoptic hypothalamic nucleus damage (Madeira et al., 1993), may have also contributed to the low milk production (Dahlborn et al., 1990). These neurotoxic effects are probably dose - dependent, since a low ethanol intake by dams (5%) failed to alter pups' body and liver weights. Despite that, we found a decrease in ATP -cit activity in the liver of all ethanol groups. Elshourbagy et al. (1990) reported that hepatic ATP -cit mRNA levels were relatively high at parturition, diminished rapidly during the suckling period (day 1± 14), and increased again after weaning in young adult animals, an effect related to the nature of the diet. In fact, lipogenesis rates in the liver are strongly affected by the composition of the diet (Pillay & Bailey, 1982); a high fat / low carbohydrate diet decreases the liver lipogenesis rate (Yeh & Zee, 1976). Hence, the decrease in ATP -cit activity found in pups' liver could be related to an increased milk lipid content in alcohol-treated rats (Vilaro et al., 1987). Accordingly, we have previously reported that 5% and 20% alcohol ingestion during lactation increased mammary gland lipogenesis rate. This is in agreement with the higher plasma level of triacylglycerols, glycerol, free fatty acids, and ketone bodies found in pups from alcohol - treated dams, as compared with those of control rats (Tavares do Carmo et al., 1999). Lipogenesis is an important pathway in the developing brain, particularly with respect to the myelination process. It is interesting that the activities of malic enzyme and ATP cit in brain were not affected by ethanol ingestion, being around 10 and 3 times higher, respectively, than in pups'
liver. Malic enzyme catalyzes the reaction of pyruvate formation from malate, with the generation of NADPH, which can be used to support biosynthetic processes during brain growth (Edmond, 1992). During lactation, a rapid brain development occurs in rats. In this period, the weight of the brain increases even more rapidly than the body weight (Dobbing, 1971). Our results showed that the intake of 10% and 20% ethanol solutions by dams decreased pups' body weight gain by 16.6% and 44.6%, respectively, in relation to the C group; however, only pups from E20 dams had a decreased brain weight (21.4%). These results elicit two hypotheses: a preservation of pups' brain or a profound reduction in body growth. The results reported here from in vitro experiments indicate that the intake of the 5% alcohol solution by dams could change brain metabolism without impairing brain growth. We have previously observed that the intake of a 5% ethanol solution by dams caused an increase in mammary gland lipogenesis rate without changing milk production, as indicated by normal pups' growth. The increase in lipid milk content could partially explain the decrease in 14 C - glucose incorporated into lipid in pups' brain slices from E5, since the lipoprotein lipase activity is high in the newborn brain (Nunez et al., 1995) and the glycerol from dietary triacylglycerols can be used for brain lipid synthesis (Custo et al., 1987). It has been demonstrated that, under normal dietary conditions, glucose -derived carbon supplies 23% of brain cholesterol synthesis, while in hypoketonemic conditions this contribution rises to almost 80% (Edmond, 1992). We have previously shown that the intake of a 20% alcohol solution by dams decreased pups' blood glucose concentration and increased pups' blood ketone bodies concentration (Tavares do Carmo et al., 1999), conditions that could decrease brain glucose consumption in vivo. However, our present results show that in the presence of an adequate glucose concentration (5 mM), there was an increase in glucose consumption and oxidation, incorporation of glucose into lipid, and lactate production from E20. It has been demonstrated that the treatment of bovine brain microvessel endothelial cells in culture with a low concentration of D - glucose (2.42 mM and 1.83 mM) for 7 days induced a significant increase in 3 -O [3H] -methyl - D -glucose uptake by these cells (Takakura et al., 1991). In the present experiments, the addition of beta - hydroxybutyrate to the incubation medium had no effect on brain glucose consumption in any group, but decreased glucose oxidation by 35% to 50%. It has been shown that, under certain conditions, the rate of ketone use can be similar to that of glucose (Singh et al., 1988). Moreover, different brain cell populations have been shown to use both glucose and ketone bodies in respiration and lipogenesis (Edmond et al., 1987; Hertz et al., 1988; Lopes- Cardozo et al., 1986). The lipogenesis from glucose was also not affected
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
by the addition of beta - hydroxybutyrate to the incubation medium. Unlike other substrates, such as lactate, hydroxybutyrate, or glutamine, glucose may also be incorporated into lipids as glycerol for glycerolipid synthesis. In fact, an important part of the incorporation of glucose into lipids by neurons or astrocytes can be accounted for by the synthesis of glycerol -borne lipid (Vicario et al., 1993). It has been reported that ethanol increases dose dependently the extracellular glutamate concentration in fetal sheep cerebral cortex (Reynolds et al., 1995). Pellerin and Magistretti (1994) have shown that glutamate uptake by astrocytes, a process mediated by activation of a Na + -dependent uptake system rather than by interaction with receptors, stimulated glucose use and lactate production from these cells. These observations could partially explain the results of increased glucose consumption and lactate production in the brain slices of E10 and E20 reported herein. The increase in lactate production could reflect a deteriorated neurological status (Therrien et al., 1991). Additionally, it has been demonstrated that the lactate -induced inhibition of the cytosolic malate ±aspartate shuttle plays a role in the synthesis of some neurotransmitters, such as glutamate and aspartate (McKenna et al., 1990; Palaiologos et al., 1988; Palaiologos et al., 1989). In accordance with our present results, McKenna et al. (1993) have shown that the inhibition of the malate ± aspartate shuttle induced a fourfold increase in glucose oxidation. From the data reported in this work, it can be concluded that the effects of maternal alcohol intake on pups' development and metabolism are dose - dependent. High ethanol intake (10% and 20%) decreased pups' body weight gain and liver weight and also affected brain glucose metabolism. Although the low intake of ethanol (5%) failed to alter pups' body weight gain and brain and liver weight, it affected brain glucose metabolism. Further work is necessary to better understand the mechanisms responsible for these effects of ethanol.
Acknowledgments The authors thank E.B. Ribeiro for reviewing the manuscript. The work was supported by CNPq and Fapesp. References Bonthius, D. J., & West, J. R. (1988). Blood alcohol concentration and microencephaly: a dose ± response study in the neonatal rat. Teratol 37, 223 ± 231. Breese, C. R., D'Costa, A., & Sonntag, W. E. (1994). Effect of in utero ethanol exposure on the postnatal ontogeny of insulin - like growth factor - 1, and type - 1 and type - 2 insulin - like growth factor receptors in the rat brain. Neuroscience 63, 579 ± 589. Corrigan, A. P., & Rider, C. C. (1983). Multiple chromatographic forms of ATP citrate lyase from rat liver. Biochem J 214, 299 ± 307.
205
Custo, G., Corazzi, L., Mastrofini, P., & Arienti, G. (1987). Glycerol incorporation into brain lipids in rat pups born to ethanol - intoxicated dams. Neurochem Res 12, 469 ± 473. Dahlborn, K., Hilali, J. H., & Rodriguez - Martinez, H. (1990). Effects of dehydration and arginine vasopressin infusions on the production of milk and the morphology of the goat udder. J Dairy Res 57, 479 ± 487. Dobbing, J. (1971). Undernutrition and the developing brain: the use of animal models to elucidate the human problem. Psychiatr Neurol Neurochir 74, 433 ± 442. Doyle, P., Cusin, I., Rohner - Jeanrenaud, F., & Jeanrenaud, B. (1995). Fourday hyperinsulinemia in euglycemic conditions alters local cerebral glucose utilization in specific brain nuclei of freely moving rats. Brain Res 684, 47 ± 55. Dubowski, K. M. (1962). An o - toluidine method for body - fluid glucose determination. Clin Chem 8, 215 ± 235. Edmond, J. (1992). Energy metabolism in developing brain cells. Can J Physiol Pharmacol 70, S118 ± S129. Edmond, J., Robbins, R. A., Bergstrom, J. D., Cole, R. A., & de Vellis, J. (1987). Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture. J Neurosci Res 18, 551 ± 561. Elshourbagy, N. A., Near, J. C., Kmetz, P. J., Sathe, G. M., Southan, C., Strickler, J. E., Gross, M., Young, J. F., Wells, T. N., & Groot, P. H. (1990). Rat ATP citrate - lyase. Molecular cloning and sequence analysis of a full - length cDNA and mRNA abundance as a function of diet, organ, and age. J Biol Chem 265, 1430 ± 1435. Engel, P. C., & Jones, J. B. (1978). Causes and elimination of erratic blanks in enzymatic metabolite assays involving the use of NAD + in alkaline hydrazine buffers: improved conditions for the assay of L - glutamate, L - lactate, and other metabolites. Anal Biochem 88, 475 ± 484. Fando, J. L., CaleÂs, C., Azuara, C., AlcaÂzar, A., & Salinas, M. (1988). Sintesis de proteinas durante el desarrollo cerebral: su regulacion. In E. Herrera (Ed.), BioquõÂmica Perinatal (Aspectos BaÂsicos e PatoloÂgicos) ( pp. 385 ± 404). Madrid: FundacioÂn RamoÂn Areces. Funchs, A. R. (1969). Ethanol and the inhibition of oxytocin release in lactating rats. Acta Endocrinol 62, 546 ± 554. Guerri, C., & Sanchis, R. (1986). Alcohol and acetaldehyde in rat's milk following ethanol administration. Life Sci 38, 1543 ± 1556. Hertz, L., Drejer, J., & Schousboe, A. (1988). Energy metabolism in glutamatergic neurons, GABAergic neurons and astrocytes in primary cultures. Neurochem Res 13, 605 ± 610. Lancaster, F. E., Mayur, B. K., Patsalos, P. N., Samorajski, T., & Wiggins, R. C. (1982). The synthesis of myelin and brain subcellular membrane proteins in the offspring of rats fed ethanol during pregnancy. Brain Res 235, 105 ± 113. Lopes - Cardozo, M., Larsson, O. M., & Schousboe, A. (1986). Acetoacetate and glucose as lipid precursors and energy substrates in primary cultures of astrocytes and neurons from mouse cerebral cortex. J Neurochem 46, 773 ± 778. Lowry, O. H., Rosenbrought, N. J., Fair, A. L., & Randall, R. J. (1951). Protein measurement with the folin ± phenol reagent. J Biol Chem 193, 265 ± 275. Madeira, M. D., Souza, N., Lieberman, A. R., & Paula-Barbosa, M. M. (1993). Effects of chronic ethanol consumption and of dehydration on the supraoptic nucleus of adult male and female rats. Neuroscience 50, 657 ± 672. McKenna, M. C., Tildon, J. T., Couto, R., Stevenson, J. H., & Caprio, F. J. (1990). The metabolism of malate by cultured rat brain astrocytes. Neurochem Res 15, 1211 ± 1220. McKenna, M. C., Tildon, J. T., Stevenson, J. H., Boatright, R., & Huang, S. (1993). Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate. Dev Neurosci 15, 320 ± 329. Newsholme, E. A., & Williams, T. (1978). The role of phosphoenolpyruvate carboxykinase in amino acid metabolism in muscle. Biochem J 176, 623 ± 626.
206
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
Nunez, M., Peinado - Onsurbe, J., VilaroÂ, S., & Llobera, M. (1995). Lipoprotein lipase activity in developing rat brain areas. Biol Neonate 68, 119 ± 127. Palaiologos, G., Hertz, L., & Schousboe, A. (1988). Evidence that aspartate aminotransferase activity and ketodicarboxylate carrier function are essential for biosynthesis of transmitter glutamate. J Neurochem 51, 317 ± 320. Palaiologos, G., Hertz, L., & Schousboe, A. (1989). Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons. Neurochem Res 14, 359 ± 366. Pellerin, L., & Magistretti, P. J. (1994). Glutamate uptake into astrocytes stimulates aerobic glycolysis: A mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci 91, 10625 ± 10629. Pillay, D., & Bailey, E. (1982). Lipogenesis at the suckling ± weaning transition in liver and brown adipose tissue of the rat. Biochim Biophys Acta 713, 663 ± 669. Rawat, A. K. (1975). Ribosomal proteins synthesis in the fetal and neonatal rat brain as influenced by maternal ethanol consumption. Res Comm Chem Path Pharmacol 12, 723 ± 732. Reinis, S., & Golman, J. M. (1980). The Development of the Brain: Biological and Functional Perspective. In C.C. Thomas (Ed.). Springfield, IL. Reynolds, J. D., Penning, D. H., Dexter, F., Atkins, B., Hrdy, J., Poduska, D., Chestnut, D. H., & Brien, J. F. (1995). Dose - dependent effects of acute in vivo ethanol exposure on extracellular glutamate concentration in the cerebral cortex of the near - term fetal sheep. Alcohol Clin Exp Res 19, 1447 ± 1453. Singh, S. P., Pullen, G. L., & Snyder, A. K. (1988). Effects of ethanol on fetal fuels and brain growth in rats. J Lab Clin Med 112, 704 ± 710. Singh, S. P., Pullen, G. L., Srivenugopal, K. S., Yuan, X. H., & Snyder, A.
K. (1992). Decreased glucose transporter 1 gene expression and glucose uptake in fetal brain exposed to ethanol. Life Sci 51, 527 ± 536. Subramanian, M. G. (1995). Effects of chronic alcohol administration on lactational performance in the rat. Alcohol 12, 137 ± 143. Subramanian, M. G., Chen, X. G., Bergeski, B. A., & Savoy - Moore, R. T. (1991). Alcohol inhibition of suckling - induced prolactin release in lactating rats: threshold evaluation. Alcohol 8, 203 ± 206. Takakura, Y., Kuentzel, S. L., Raub, T. J., Davies, A., Baldwin, S. A., & Borchardt, R. T. (1991). Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. I. Basic characteristics and effects of D - glucose and insulin. Biochim Biophys Acta 1070, 1 ± 10. Tavares - do - Carmo, M. G., & Nascimento - Curi, C. M. (1990). Effect of ethanol intake during lactation on the metabolism of dams and on pup development. Braz J Med Biol Res 23, 1161 ± 1163. Tavares do Carmo, M. G., Oller do Nascimento, C. M., Martin, A., & Herrera, E. (1999). Ethanol intake during lactation impairs milk production in rats and affects growth and metabolism of suckling pups. Alcohol 18, 71 ± 76. Therrien, G., Giguere, J. F., & Butterworth, R. F. (1991). Increased cerebrospinal fluid lactate reflects deterioration of neurological status in experimental portal - systemic encephalopathy. Metab Brain Dis 6, 225 ± 231. Vicario, C., Tabernero, A., & Medina, J. M. (1993). Regulation of lactate metabolism by albumin in rat neurons and astrocytes from primary culture. Pediatr Res 34, 709 ± 715. VilaroÂ, S., Vinas, O., Remesar, X., & Herrera, E. (1987). Effects of chronic ethanol consumption on lactational performance in rat: mammary gland and milk composition and pups' growth and metabolism. Pharmacol Biochem Behav 27, 333 ± 339. Yeh, Y. Y., & Zee, P. (1976). Relation of ketosis to metabolic changes induced by acute medium - chain triglyceride feeding in rats. J Nutr 106, 58 ± 67.
Ethanol intake during lactation II. Effects on pups' liver and brain metabolism L.M. Oyamaa, R.C. Coutob, G.E.C. Coutoc, A.R. DaÃmasod, C.M. Oller do Nascimentoa,* a
Departamento de Fisiologia, Universidade Federal de SaÄo Paulo - EPM, SaÄo Paulo, SP 04023 - 060, Brazil b Departamento de CieÃncias FisioloÂgicas, Universidade do Amazonas, Manaus, AM, 69007 - 000, Brazil c Departamento de Desporto e Atividades ComunitaÂrias, Universidade do Amazonas, Manaus, AM, 69007 - 000, Brazil d Departamento de EducacËaÄo FõÂsica e Motricidade Humana, Universidade Federal de SaÄo Carlos, SaÄo Carlos, SP, 13565 - 535, Brazil Received 28 July 1999; received in revised form 29 November 1999; accepted 13 December 1999
Abstract Lactating rats, with litters adjusted to 8 pups on day 1, were divided into 4 groups: control animals (C), which received water and Nuvilab chow ad libitum, and ethanol animals (E), which received 20% (E20), 10% (E10), or 5% (E5) ethanol diluted in the drinking water and Nuvilab chow ad libitum. On day 12 of life, the pups were weighed and decapitated. The intake of 10% and 20% ethanol solutions by the lactating rats decreased the pups' body weight and liver weight. The pups' liver ATP - citrate lyase activity was decreased in all ethanol groups. The pups' brain weight decreased in E20 only. Glucose metabolism and lactate production were studied in the pups' brain slices, which were incubated at 37°C in Krebs ± Henseleit buffer under carbogen in the presence of glucose (5 mM) plus 14C - glucose (0.04 mCi) with or without beta - hydroxybutyrate or insulin. Study of the incubated pups' brain slices showed that the intake of the 20% ethanol solution by the dams increased glucose consumption, oxidation, lactate production, and lipogenesis rate from glucose in all media studied, as compared with findings in the C group. In the pups' brain slices, the lactate production and lipogenesis rate from glucose were higher in E10 than in the C group. The addition of beta - hydroxybutyrate to the incubation medium caused a decrease in glucose oxidation in C, E5, and E20 and an increase in glucose consumption in E10. Ingestion of the 5% ethanol solution by dams decreased the pups' brain lipogenesis rate from glucose in all media studied. We concluded that the effects of maternal alcohol intake on the pups' development and metabolism are dose - dependent. High amounts of ethanol intake (10% or 20%) caused a great impairment in the pups' growth, as well as their liver and brain metabolism. The low dose (5%) did not affect the pups' body weight gain or their brain and liver weight, but it did alter brain glucose metabolism. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Alcohol; Lactation; Pups' growth; Liver; Brain; Rat
1. Introduction There has been extensive documentation that alcohol consumption during pregnancy has a detrimental effect on fetal growth and development and alters hepatic and cerebral intermediary metabolism, neurotransmitters, and lipid metabolism (Breese et al., 1994; Lancaster et al., 1982; Rawat, 1975). During lactation, the ingestion of a diet that obtained approximately one third of its calories from ethanol reduced * Corresponding author. Departamento de Fisiologia Ð Disciplina de Neurofisiologia e Fisiologia EndoÂcrina, Rua Botucatu, 862, 2° andar EdifõÂcio de CieÃncias BiomeÂdicas, Vila Clementino Ð SaÄo Paulo, SP 04023 - 060, Brazil. Tel.: +55 - 11 - 576 - 4527; fax: +55 - 11 - 570 - 7675. E-mail address: [email protected] (C.M. Oller do Nascimento).
milk production, altered milk composition (Vilaro et al., 1987), and retarded offsprings' body and central nervous system growth (Tavares -do - Carmo & Nascimento - Curi, 1990). It has also been demonstrated that alcohol administration to lactating rats inhibited suckling -induced prolactin release (Subramanian et al., 1991). Ingestion of ethanol during lactation resulted in the presence of ethanol and acetaldehyde in the milk (Guerri & Sanchis, 1986). Recently, we have shown that the effect of ethanol on dams' and pups' metabolism appears to be related to the dose of alcohol administered. High doses of ethanol (10% and 20%) ingested by the lactating rats promoted modifications in dams' metabolism and impaired pups' growth, while the intake of a low ethanol dose (5%) did not affect pups' growth despite changing dams' metabolism. In addition, it has been reported that pups receiving
0741-8329/00/$ ± see front matter D 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 9 9 ) 0 0 0 7 4 - 4
202
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
Table 1 Effect of ethanol intake by lactating rats on pups liver weight (g), protein content (mg / g), ATP - cit, and malic enzyme activities (mmol / min / 100 mg of protein) and on pups' body weight (g) day 12 after birth (g) C E5 E10 E20
Liver weight
Protein
0.59 0.03 0.55 0.02 0.36 0.02 0.29 0.01
133.4 6.76 142.45 4.56 139.33 7.18 145.04 4.88
(6) (6) (6)* (6)*
(9) (17) (16) (18)
Malic enzyme
ATP - cit
0.03 0.007 (9) 0.06 0.01 (12) 0.06 0.005 (13) 0.06 0.007 (13)
0.18 0.03 0.13 0.02 0.12 0.01 0.08 0.01
Body weight (7) (13)* (13)* (16)*
19.57 0.42 (10) 20.02 0.6 (10) 16.32 0.69 (10)* 10.83 0.51 (10)*
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
received 20% (E20), 10% (E10), or 5% (E5) ethanol solutions diluted in the drinking water and Nuvilab chow ad libitum. The mean of dams' ethanol intake (ml /100 g b.w.) on day 12 of lactation in E5, E10, and E20 was 1.38 0.08, 1.94 0.10, and 2.86 0.10, which resulted in 2.87 1.06, 43.45 11.50, and 100.66 25.30 blood ethanol concentration (mg / dl), respectively. On day 12 of life the pups were weighed and decapitated. The liver and the brain were removed, weighed, and stored in a deep freezer (ÿ70°C) for posterior analysis.
4.0 g/ kg/day of ethyl - alcohol did not exhibit significant microencephaly when compared with controls (Bonthius & West, 1988). Prenatal ethanol exposure decreases the content of neuronal glucose transporter mRNA, which would inhibit the ability of the fetal brain to take up glucose (Singh et al., 1992). In rats, a high degree of brain development and myelination occurs during the first 15 days after birth (Fando et al., 1988; Reinis & Golman, 1980). Findings from many studies from 1970 ± 1980, both in human infants and in the rat pup model, showed that the glucose and ketone bodies acetoacetate and D -(ÿ)- 3- hydroxybutyrate are taken up by the brain and used for energy production and as carbon sources for lipogenesis (Edmond, 1992). The aim of the present study was to evaluate the effect of different amounts of maternal ethanol ingestion (5%, 10%, or 20%) during lactation on pups' liver and brain metabolism. Glucose consumption and oxidation, lactate production, and lipid synthesis by pups' brain slices in vitro were also determined.
2.2. Tissue ATP -citrate lyase and malic enzymes activities For measurement of ATP -citrate lyase (ATP - cit) and malic enzymes activities, the liver and brain were homogenized at a proportion of 1:5, with the extraction medium appropriate for each enzyme, and activities were measured as described previously by Corrigan and Rider (1983) and Newsholme and Williams (1978), respectively. Tissue protein concentration was determined by the method used by Lowry et al. (1951).
2. Methods
2.3. Brain incubation
The Experimental Research Committee of the Federal Sao Paulo University approved all procedures involving animals.
Samples of brain ( 100 mg) were sliced for the incubation procedure. Incubations were performed at 37°C in 25 ml Erlenmeyer flasks equipped with a central well. The incubation medium consisted of 2.0 ml of Krebs ±Henseleit buffer containing 5 mM of glucose plus 0.04 mCi of 14C glucose. Eleven micromolar beta - hydroxybutyrate, an important energy supplier and a carbon source for lipogenesis in the developing brain (Edmond, 1992), or 0.5 U / ml insulinÐsince it has been previously demonstrated that chronic hyperinsulinemia could alter glucose utilization by several brain regions (Doyle et al., 1995) Ð was added to
2.1. Animals and procedures Wistar lactating rats were housed under constant conditions of lighting (12 h/ 12 h light/dark) and temperature (24 1°C) in individual cages with 8 pups each. On the first day postpartum, lactating dams were divided into four groups: control animals (C), which received water and Nuvilab chow ad libitum, and ethanol animals, which
Table 2 Effect of ethanol intake by lactating rats on pups' brain weight (g), protein content (mg / g), ATP - cit, and malic enzyme activities (mmol / min / 100 mg of protein) C E5 E10 E20
Weight
Protein
1.03 0.015 (6) 1.02 0.02 (6) 0.95 0.02 (6) 0.81 0.04 (6)*
66.87 4.83 72.58 2.40 64.85 4.70 64.54 4.39
(9) (10) (10) (10)
Malic enzyme
ATP - cit
0.60 0.04 (9) 0.53 0.01 (10) 0.62 0.006 (10) 0.58 0.04 (9)
0.47 0.05 0.35 0.03 0.53 0.07 0.39 0.03
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
(7) (10) (9) (9)
L.M. Oyama et al. / Alcohol 21 (2000) 201±206 Table 3 Effect of ethanol intake by lactating rats on pups' brain glucose consumption (mmol / min / 100 mg of tissue / h) Glucose C E5 E10 E20
25.63 1.65 26.9 1.88 25.76 1.56 33.29 2.19
(9) (6) (8) (10)*
Glucose + b - hydroxybutyrate
Glucose + insulin
22.05 1.87 23.42 1.88 29.01 2.74 30.57 1.97
23.82 1.43 25.34 1.70 32.27 2.94 36.22 1.74
(10) (9) (8)* (12)*
(11) (9) (8)* (8)*
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
the medium. The flasks were continuously shaken and flushed with carbogenium (O2CO2, 95%/5%) during the incubation period. After 1 h, incubation was stopped by the addition of 0.5 ml of 4N H2SO4 into the main well. Three tenths milliliter of NaOH (1N) was added to the central well for 14CO2 collection, during 2 h after the incubation was stopped. After that, brain slices were removed, and total lipids were extracted in 10 ml of chloroform ±methanol (2:1), the radioactivity in this extract representing the conversion of [1- 14C]- glucose into lipid. Rates of conversion of [1 - 14C] -glucose into 14CO2 and incorporation into the lipid fraction were expressed as mmol /h g of tissue. The incubation medium used for determination of brain glucose consumption and lactate production was the same as that used by Dubowski (1962) and Engel and Jones (1978), respectively. 2.4. Statistical analysis Results were expressed as mean SEM, and statistical comparison among groups was made by one- way analysis of variance. Differences among means were tested for significance by the Duncan's multiple range test, and p < 0.05 was taken as the level of significance. 3. Results As shown in Table 1, pups' body and liver weights decreased in E10 and E20 when compared with findings Table 4 Effect of ethanol intake by lactating rats on pups' brain glucose oxidation (mmol / g tissue / h) Glucose C E5 E10 E20
0.66 0.11 (11) 0.63 0.08 (6) 0.67 0.09 (9) 1.08 0.10 (13)*
Glucose + b - hydroxybutyrate 0.33 0.07 0.31 0.04 0.43 0.06 0.64 0.09
y
(12) (5)y (11) (13)*,y
Table 5 Effect of ethanol intake by lactating rats on pups' brain lipogenesis rate (14C - glucose incorporated in lipid / g tissue / h) Glucose C E5 E10 E20
(13) (5) (8) (14)*
Values is the mean SEM. The number of rats used in each determination are shown in parentheses. y Significantly different from glucose medium at p < 0.05. * Significantly different from control group at p < 0.05.
1.24 0.13 0.66 0.05 2.05 0.17 1.84 0.12
(12) (9)* (10)* (10)*
Glucose + b - hydroxybutyrate
Glucose + insulin
1.15 0.11 (13) 0.71 0.04 (10)* 1.53 0.16 (11)*,y 1.96 0.10 (8)*
1.44 0.14 0.89 0.09 1.57 0.16 1.79 0.16
(15) (12)* (12)y (7)
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05. y Significantly different from glucose medium at p < 0.05.
in the C group. However, the ingestion of different ethanol concentrations by the lactating rats did not alter protein content and malic enzyme activity in this tissue. On the other hand, the ATP -cit activity of pups' liver was decreased in all experimental groups. No changes were observed in protein content, nor in malic or ATP - cit enzyme activities, in pup's brain from all experimental groups, though brain weight was lower in E20 than in the C group (Table 2). Study of the incubated pups' brain slices showed that ingestion of the 20% ethanol solution by the lactating rats increased glucose consumption in all media studied, as compared with findings in the C group. When beta -hydroxybutyrate or insulin was added to the incubation medium, the consumption of glucose by the brain of E10 became higher than that of the C group (Table 3). Glucose oxidation increased in E20 when compared with that of the C group. Addition of beta -hydroxybutyrate to the incubation medium caused a decrease in glucose oxidation in C, E5, and E20. No alteration in this parameter was observed in E10 (Table 4). Ingestion of the 5% ethanol solution by the dams decreased pups' brain lipogenesis rate from glucose in all media studied. The 10% and 20% ethanol solutions increased this parameter in the presence or absence of beta hydroxybutyrate when compared with findings in the C group. In E10, the lipogenesis rate from glucose was higher when the medium contained only glucose, in relation to the other media (Table 5).
Table 6 Effect of ethanol intake by lactating rats on pups' brain lactate production (mmol / g of tissue / h)
Glucose + insulin 0.43 0.09 0.48 0.05 0.55 0.09 1.19 0.10
203
Glucose C E5 E10 E20
2.21 0.20 2.60 0.17 3.04 0.14 4.07 0.30
(11) (11) (10)* (6)*
Glucose + b - hydroxybutyrate
Glucose + insulin
2.58 0.29 2.77 0.13 2.80 0.12 3.65 0.12
2.20 0.21 2.35 0.16 2.72 0.16 4.26 0.22
(10) (8) (10) (7)*
(11) (10) (11) (7)*
Values are the mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from control group at p < 0.05.
204
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
Lactate production was increased in E20 in all studied media. In E10, lactate production was higher than that in the C group in the medium containing only glucose (Table 6). 4. Discussion The results obtained in this study showed that maternal consumption of ethanol during lactation resulted in serious modifications in pups' nutritional status and metabolism. These effects were related to the amount of ethanol ingested by dams. Our results show that the intake of 10% and 20% ethanol solutions by dams decreased pups' body weight and liver weight. The growth and development of pups are directly related to the quantity and quality of the milk. It has been previously shown that the decrease in milk production induced by a high ethanol intake during lactation could not be attributed only to the undernourishing effect of ethanol (Tavares do Carmo et al., 1999). This could be related to the fact that alcohol administration during lactation affects milk consumption by rat pups, by decreasing suckling -induced prolactin and oxytocin release (Funchs, 1969; Subramanian, 1995). Furthermore, ethanol neurotoxicity, which leads to dehydration and hyperosmolarity due to supraoptic hypothalamic nucleus damage (Madeira et al., 1993), may have also contributed to the low milk production (Dahlborn et al., 1990). These neurotoxic effects are probably dose - dependent, since a low ethanol intake by dams (5%) failed to alter pups' body and liver weights. Despite that, we found a decrease in ATP -cit activity in the liver of all ethanol groups. Elshourbagy et al. (1990) reported that hepatic ATP -cit mRNA levels were relatively high at parturition, diminished rapidly during the suckling period (day 1± 14), and increased again after weaning in young adult animals, an effect related to the nature of the diet. In fact, lipogenesis rates in the liver are strongly affected by the composition of the diet (Pillay & Bailey, 1982); a high fat / low carbohydrate diet decreases the liver lipogenesis rate (Yeh & Zee, 1976). Hence, the decrease in ATP -cit activity found in pups' liver could be related to an increased milk lipid content in alcohol-treated rats (Vilaro et al., 1987). Accordingly, we have previously reported that 5% and 20% alcohol ingestion during lactation increased mammary gland lipogenesis rate. This is in agreement with the higher plasma level of triacylglycerols, glycerol, free fatty acids, and ketone bodies found in pups from alcohol - treated dams, as compared with those of control rats (Tavares do Carmo et al., 1999). Lipogenesis is an important pathway in the developing brain, particularly with respect to the myelination process. It is interesting that the activities of malic enzyme and ATP cit in brain were not affected by ethanol ingestion, being around 10 and 3 times higher, respectively, than in pups'
liver. Malic enzyme catalyzes the reaction of pyruvate formation from malate, with the generation of NADPH, which can be used to support biosynthetic processes during brain growth (Edmond, 1992). During lactation, a rapid brain development occurs in rats. In this period, the weight of the brain increases even more rapidly than the body weight (Dobbing, 1971). Our results showed that the intake of 10% and 20% ethanol solutions by dams decreased pups' body weight gain by 16.6% and 44.6%, respectively, in relation to the C group; however, only pups from E20 dams had a decreased brain weight (21.4%). These results elicit two hypotheses: a preservation of pups' brain or a profound reduction in body growth. The results reported here from in vitro experiments indicate that the intake of the 5% alcohol solution by dams could change brain metabolism without impairing brain growth. We have previously observed that the intake of a 5% ethanol solution by dams caused an increase in mammary gland lipogenesis rate without changing milk production, as indicated by normal pups' growth. The increase in lipid milk content could partially explain the decrease in 14 C - glucose incorporated into lipid in pups' brain slices from E5, since the lipoprotein lipase activity is high in the newborn brain (Nunez et al., 1995) and the glycerol from dietary triacylglycerols can be used for brain lipid synthesis (Custo et al., 1987). It has been demonstrated that, under normal dietary conditions, glucose -derived carbon supplies 23% of brain cholesterol synthesis, while in hypoketonemic conditions this contribution rises to almost 80% (Edmond, 1992). We have previously shown that the intake of a 20% alcohol solution by dams decreased pups' blood glucose concentration and increased pups' blood ketone bodies concentration (Tavares do Carmo et al., 1999), conditions that could decrease brain glucose consumption in vivo. However, our present results show that in the presence of an adequate glucose concentration (5 mM), there was an increase in glucose consumption and oxidation, incorporation of glucose into lipid, and lactate production from E20. It has been demonstrated that the treatment of bovine brain microvessel endothelial cells in culture with a low concentration of D - glucose (2.42 mM and 1.83 mM) for 7 days induced a significant increase in 3 -O [3H] -methyl - D -glucose uptake by these cells (Takakura et al., 1991). In the present experiments, the addition of beta - hydroxybutyrate to the incubation medium had no effect on brain glucose consumption in any group, but decreased glucose oxidation by 35% to 50%. It has been shown that, under certain conditions, the rate of ketone use can be similar to that of glucose (Singh et al., 1988). Moreover, different brain cell populations have been shown to use both glucose and ketone bodies in respiration and lipogenesis (Edmond et al., 1987; Hertz et al., 1988; Lopes- Cardozo et al., 1986). The lipogenesis from glucose was also not affected
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
by the addition of beta - hydroxybutyrate to the incubation medium. Unlike other substrates, such as lactate, hydroxybutyrate, or glutamine, glucose may also be incorporated into lipids as glycerol for glycerolipid synthesis. In fact, an important part of the incorporation of glucose into lipids by neurons or astrocytes can be accounted for by the synthesis of glycerol -borne lipid (Vicario et al., 1993). It has been reported that ethanol increases dose dependently the extracellular glutamate concentration in fetal sheep cerebral cortex (Reynolds et al., 1995). Pellerin and Magistretti (1994) have shown that glutamate uptake by astrocytes, a process mediated by activation of a Na + -dependent uptake system rather than by interaction with receptors, stimulated glucose use and lactate production from these cells. These observations could partially explain the results of increased glucose consumption and lactate production in the brain slices of E10 and E20 reported herein. The increase in lactate production could reflect a deteriorated neurological status (Therrien et al., 1991). Additionally, it has been demonstrated that the lactate -induced inhibition of the cytosolic malate ±aspartate shuttle plays a role in the synthesis of some neurotransmitters, such as glutamate and aspartate (McKenna et al., 1990; Palaiologos et al., 1988; Palaiologos et al., 1989). In accordance with our present results, McKenna et al. (1993) have shown that the inhibition of the malate ± aspartate shuttle induced a fourfold increase in glucose oxidation. From the data reported in this work, it can be concluded that the effects of maternal alcohol intake on pups' development and metabolism are dose - dependent. High ethanol intake (10% and 20%) decreased pups' body weight gain and liver weight and also affected brain glucose metabolism. Although the low intake of ethanol (5%) failed to alter pups' body weight gain and brain and liver weight, it affected brain glucose metabolism. Further work is necessary to better understand the mechanisms responsible for these effects of ethanol.
Acknowledgments The authors thank E.B. Ribeiro for reviewing the manuscript. The work was supported by CNPq and Fapesp. References Bonthius, D. J., & West, J. R. (1988). Blood alcohol concentration and microencephaly: a dose ± response study in the neonatal rat. Teratol 37, 223 ± 231. Breese, C. R., D'Costa, A., & Sonntag, W. E. (1994). Effect of in utero ethanol exposure on the postnatal ontogeny of insulin - like growth factor - 1, and type - 1 and type - 2 insulin - like growth factor receptors in the rat brain. Neuroscience 63, 579 ± 589. Corrigan, A. P., & Rider, C. C. (1983). Multiple chromatographic forms of ATP citrate lyase from rat liver. Biochem J 214, 299 ± 307.
205
Custo, G., Corazzi, L., Mastrofini, P., & Arienti, G. (1987). Glycerol incorporation into brain lipids in rat pups born to ethanol - intoxicated dams. Neurochem Res 12, 469 ± 473. Dahlborn, K., Hilali, J. H., & Rodriguez - Martinez, H. (1990). Effects of dehydration and arginine vasopressin infusions on the production of milk and the morphology of the goat udder. J Dairy Res 57, 479 ± 487. Dobbing, J. (1971). Undernutrition and the developing brain: the use of animal models to elucidate the human problem. Psychiatr Neurol Neurochir 74, 433 ± 442. Doyle, P., Cusin, I., Rohner - Jeanrenaud, F., & Jeanrenaud, B. (1995). Fourday hyperinsulinemia in euglycemic conditions alters local cerebral glucose utilization in specific brain nuclei of freely moving rats. Brain Res 684, 47 ± 55. Dubowski, K. M. (1962). An o - toluidine method for body - fluid glucose determination. Clin Chem 8, 215 ± 235. Edmond, J. (1992). Energy metabolism in developing brain cells. Can J Physiol Pharmacol 70, S118 ± S129. Edmond, J., Robbins, R. A., Bergstrom, J. D., Cole, R. A., & de Vellis, J. (1987). Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture. J Neurosci Res 18, 551 ± 561. Elshourbagy, N. A., Near, J. C., Kmetz, P. J., Sathe, G. M., Southan, C., Strickler, J. E., Gross, M., Young, J. F., Wells, T. N., & Groot, P. H. (1990). Rat ATP citrate - lyase. Molecular cloning and sequence analysis of a full - length cDNA and mRNA abundance as a function of diet, organ, and age. J Biol Chem 265, 1430 ± 1435. Engel, P. C., & Jones, J. B. (1978). Causes and elimination of erratic blanks in enzymatic metabolite assays involving the use of NAD + in alkaline hydrazine buffers: improved conditions for the assay of L - glutamate, L - lactate, and other metabolites. Anal Biochem 88, 475 ± 484. Fando, J. L., CaleÂs, C., Azuara, C., AlcaÂzar, A., & Salinas, M. (1988). Sintesis de proteinas durante el desarrollo cerebral: su regulacion. In E. Herrera (Ed.), BioquõÂmica Perinatal (Aspectos BaÂsicos e PatoloÂgicos) ( pp. 385 ± 404). Madrid: FundacioÂn RamoÂn Areces. Funchs, A. R. (1969). Ethanol and the inhibition of oxytocin release in lactating rats. Acta Endocrinol 62, 546 ± 554. Guerri, C., & Sanchis, R. (1986). Alcohol and acetaldehyde in rat's milk following ethanol administration. Life Sci 38, 1543 ± 1556. Hertz, L., Drejer, J., & Schousboe, A. (1988). Energy metabolism in glutamatergic neurons, GABAergic neurons and astrocytes in primary cultures. Neurochem Res 13, 605 ± 610. Lancaster, F. E., Mayur, B. K., Patsalos, P. N., Samorajski, T., & Wiggins, R. C. (1982). The synthesis of myelin and brain subcellular membrane proteins in the offspring of rats fed ethanol during pregnancy. Brain Res 235, 105 ± 113. Lopes - Cardozo, M., Larsson, O. M., & Schousboe, A. (1986). Acetoacetate and glucose as lipid precursors and energy substrates in primary cultures of astrocytes and neurons from mouse cerebral cortex. J Neurochem 46, 773 ± 778. Lowry, O. H., Rosenbrought, N. J., Fair, A. L., & Randall, R. J. (1951). Protein measurement with the folin ± phenol reagent. J Biol Chem 193, 265 ± 275. Madeira, M. D., Souza, N., Lieberman, A. R., & Paula-Barbosa, M. M. (1993). Effects of chronic ethanol consumption and of dehydration on the supraoptic nucleus of adult male and female rats. Neuroscience 50, 657 ± 672. McKenna, M. C., Tildon, J. T., Couto, R., Stevenson, J. H., & Caprio, F. J. (1990). The metabolism of malate by cultured rat brain astrocytes. Neurochem Res 15, 1211 ± 1220. McKenna, M. C., Tildon, J. T., Stevenson, J. H., Boatright, R., & Huang, S. (1993). Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate. Dev Neurosci 15, 320 ± 329. Newsholme, E. A., & Williams, T. (1978). The role of phosphoenolpyruvate carboxykinase in amino acid metabolism in muscle. Biochem J 176, 623 ± 626.
206
L.M. Oyama et al. / Alcohol 21 (2000) 201±206
Nunez, M., Peinado - Onsurbe, J., VilaroÂ, S., & Llobera, M. (1995). Lipoprotein lipase activity in developing rat brain areas. Biol Neonate 68, 119 ± 127. Palaiologos, G., Hertz, L., & Schousboe, A. (1988). Evidence that aspartate aminotransferase activity and ketodicarboxylate carrier function are essential for biosynthesis of transmitter glutamate. J Neurochem 51, 317 ± 320. Palaiologos, G., Hertz, L., & Schousboe, A. (1989). Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons. Neurochem Res 14, 359 ± 366. Pellerin, L., & Magistretti, P. J. (1994). Glutamate uptake into astrocytes stimulates aerobic glycolysis: A mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci 91, 10625 ± 10629. Pillay, D., & Bailey, E. (1982). Lipogenesis at the suckling ± weaning transition in liver and brown adipose tissue of the rat. Biochim Biophys Acta 713, 663 ± 669. Rawat, A. K. (1975). Ribosomal proteins synthesis in the fetal and neonatal rat brain as influenced by maternal ethanol consumption. Res Comm Chem Path Pharmacol 12, 723 ± 732. Reinis, S., & Golman, J. M. (1980). The Development of the Brain: Biological and Functional Perspective. In C.C. Thomas (Ed.). Springfield, IL. Reynolds, J. D., Penning, D. H., Dexter, F., Atkins, B., Hrdy, J., Poduska, D., Chestnut, D. H., & Brien, J. F. (1995). Dose - dependent effects of acute in vivo ethanol exposure on extracellular glutamate concentration in the cerebral cortex of the near - term fetal sheep. Alcohol Clin Exp Res 19, 1447 ± 1453. Singh, S. P., Pullen, G. L., & Snyder, A. K. (1988). Effects of ethanol on fetal fuels and brain growth in rats. J Lab Clin Med 112, 704 ± 710. Singh, S. P., Pullen, G. L., Srivenugopal, K. S., Yuan, X. H., & Snyder, A.
K. (1992). Decreased glucose transporter 1 gene expression and glucose uptake in fetal brain exposed to ethanol. Life Sci 51, 527 ± 536. Subramanian, M. G. (1995). Effects of chronic alcohol administration on lactational performance in the rat. Alcohol 12, 137 ± 143. Subramanian, M. G., Chen, X. G., Bergeski, B. A., & Savoy - Moore, R. T. (1991). Alcohol inhibition of suckling - induced prolactin release in lactating rats: threshold evaluation. Alcohol 8, 203 ± 206. Takakura, Y., Kuentzel, S. L., Raub, T. J., Davies, A., Baldwin, S. A., & Borchardt, R. T. (1991). Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. I. Basic characteristics and effects of D - glucose and insulin. Biochim Biophys Acta 1070, 1 ± 10. Tavares - do - Carmo, M. G., & Nascimento - Curi, C. M. (1990). Effect of ethanol intake during lactation on the metabolism of dams and on pup development. Braz J Med Biol Res 23, 1161 ± 1163. Tavares do Carmo, M. G., Oller do Nascimento, C. M., Martin, A., & Herrera, E. (1999). Ethanol intake during lactation impairs milk production in rats and affects growth and metabolism of suckling pups. Alcohol 18, 71 ± 76. Therrien, G., Giguere, J. F., & Butterworth, R. F. (1991). Increased cerebrospinal fluid lactate reflects deterioration of neurological status in experimental portal - systemic encephalopathy. Metab Brain Dis 6, 225 ± 231. Vicario, C., Tabernero, A., & Medina, J. M. (1993). Regulation of lactate metabolism by albumin in rat neurons and astrocytes from primary culture. Pediatr Res 34, 709 ± 715. VilaroÂ, S., Vinas, O., Remesar, X., & Herrera, E. (1987). Effects of chronic ethanol consumption on lactational performance in rat: mammary gland and milk composition and pups' growth and metabolism. Pharmacol Biochem Behav 27, 333 ± 339. Yeh, Y. Y., & Zee, P. (1976). Relation of ketosis to metabolic changes induced by acute medium - chain triglyceride feeding in rats. J Nutr 106, 58 ± 67.