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Ethanol intake during lactation
Alcohol 21 (2000) 195 ± 200
Ethanol intake during lactation I. Effects on dams' metabolism and pups' body weight gain 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 Wistar lactating rats (8 pups per dam) had free access to either tap water (control group, C) or one of three concentrations of ethanol (E) in the drinking water: 5% (E5), 10% (E10), and 20% (E20). All animals received normal rat chow ad libitum and were killed on day 12 of lactation. Intake of both 10% and 20% ethanol solutions decreased food intake, dams' body weight, and pups' body weight gain as compared with findings in the C group. The relative weights (g / 100g b.w.) of the mammary glands (MG) and of the parametrial white adipose tissue depot were decreased only in E20 as compared with findings in the C group. Protein and lipid content of these tissues were not altered in any of the ethanol groups. In comparison with the C group, the lipogenesis rate was increased in the MG (135.6%) and liver (120.2%) in E5 and the MG (58.1%) and parametrial white adipose tissue depot (147.0%) in E20. No modifications in lipogenesis rate were noted in E10. The malic enzyme activity was decreased in the MG in E10 (25.3%) and E20 (26.4%) and in the liver in E20 (45.7%). In E5, however, it was increased in the liver (23.9%). The activity of ATP - citrate lyase in the liver was decreased in E20 (56.7%), while it was increased by 37.5% in E5 and 34.2% in E10. Blood glucose concentration of dams was not affected by ethanol ingestion. However, plasma triacylglycerol concentration was higher in E10 (17.9%) and E20 (13.3%) than in the C group, and plasma protein was lower in E20 (15.7%) than in C. We concluded that alcohol intake during lactation increased the MG lipogenesis rate; although at the highest dose, this metabolic alteration was not enough to allow normal pups' growth. However, the low dose of ethanol (5%), despite having altered dams' metabolism, did not affect pups' body weight gain. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Alcohol; Lactation; Mammary gland; Liver; Lipogenesis; Rat; ATP - citrate lyase; Malic enzyme
1. Introduction Lactation is characterized by an increased demand for metabolic substrates to provide the constituents of milk (e.g., lactose, lipid, and protein). Adequate milk production and composition is highly dependent on several physiological adaptations, such as increased cardiac output; liver, heart, mammary gland, and intestine hypertrophy; and increased food intake (Chatwin et al., 1969; Craft, 1970; Fell et al., 1963).
* 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).
Chronic alcohol ingestion is associated with undernourishment due, at least in part, to a low food intake and decreased capacity for intestinal absorption (Lieber, 1984). In rats, maternal alcoholism during pregnancy resulted in severe prenatal growth deficiency (Testar et al., 1986). Vilaro et al. (1987) showed that chronic ethanol ingestion during gestation and lactation altered mammary gland function, lowering total milk production while increasing its lipid content. Accordingly, we have previously observed that the ingestion of a 20% ethanol solution by lactating rats decreased pups' body weight gain and altered the mammary gland lipid metabolism, as shown by an elevated rate of in vivo lipogenesis (Tavares - do- Carmo & Nascimento - Curi, 1990). On the other hand, mammary gland lipoprotein lipase activity and uptake of orally administered triacylglycerol were both diminished by the ethanol treatment during lactation (Tavares do Carmo et al., 1996).
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 3 - 2
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L.M. Oyama et al. / Alcohol 21 (2000) 195± 200
Fig. 3. Effect of ethanol intake by lactating rats on pups' body weight gain (g). The symbols are: . C; 6 E5; ~ E10; 5 E20. Values are mean SEM of 10 animals. * Significantly different from C at p < 0.05.
Fig. 1. Effect of ethanol intake by lactating rats on dams' (A) food intake (g) and (B) ethanol consumption (ml / 100 g b.w. / day). Values are mean SEM of 10 animals. * Significantly different from C at p < 0.05. + Significantly different from day 1 in the same group at p < 0.05. # Significantly different from E5 at p < 0.05.
Little et al. (1989) have demonstrated that in human beings ethanol ingested through breast milk had a slight but significant detrimental effect on motor, but not mental, development in breast - fed infants. However, a low dose of alcohol has been traditionally recommended to lactating mothers as an auxiliary factor for lactation because it
provides additional calories and fluid (Mennella & Beauchamp, 1993) and facilitates the ejection reflex (Auerbach et al., 1987). These data suggest that the safety of alcohol consumption, particularly with respect to the presence or absence of deleterious effects to lactation, may depend on the dosage consumed. However, it has been shown that low ethanol ingestion (5%) by lactating rats decreased milk retinol concentration and reduced food intake (Albuquerque et al., 1998). The aim of the present study was to examine the effect of consumption of 5%, 10%, or 20% ethanol in the drinking water during lactation on dams' lipid metabolism and its consequences to pups' body weight gain. 2. Methods The Experimental Research Committee of the Federal SaÄo Paulo University approved all procedures involving animals. 2.1. Animals and procedures Wistar lactating rats were housed in individual cages under constant conditions of lighting (12 h / 12 h) and temperature (24 1°C) and fed Nuvilab rat chow ad libitum. On the first day post partum, after adjustment of the litters to eight pups, dams were divided into four groups: a control group (C), which received drinking water ad libitum; and ethanol groups (E), which received free access to ethanol in Table 1 Effect of ethanol intake by lactating rats on tissue weight (g / 100 g)
Fig. 2. Effect of ethanol ingestion by lactating rats on dams' body weight gain (g). The symbols are: . C; 6 E5; ~ E10; 5 E20. Values are mean SEM of 10 animals. * Significantly different from C at p < 0.05. + Significantly different from day 1 in the same group at p < 0.05.
C E5 E10 E20
Liver
PAR
MG
4.11 0.09 (8) 4.30 0.16 (6) 4.09 0.13 (9) 3.95 0.14 (5)
1.57 0.19 (7) 1.44 0.22 (6) 1.55 0.08 (10) 0.80 0.21* (5)
4.58 0.27 (6) 3.96 0.15 (5) 4.27 0.22 (8) 3.21 0.12* (5)
Values are mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
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Table 2 Effect of ethanol intake by lactating rats on tissue protein and lipid content (mg / g) Protein C E5 E10 E20
Lipid
Liver
PAR
139.84 9.61 (10) 160.78 5.84 (10) 141.91 10.01 (9) 146.69 10.92 (10)
22.68 3.70 20.96 1.41 19.38 0.92 21.18 1.74
(9) (15) (14) (15)
MG
Liver
97.0 4.58 (10) 114.0 4.53 (10) 98.53 7.18 (10) 102.86 4.62 (10)
3.81 0.15 3.36 0.07 3.88 0.09 4.10 0.20
the drinking water in the concentration of 5% (E5), 10% (E10), or 20% (E20). Daily food and ethanol intakes and body weights were measured throughout the treatment period. Pups were weighed every other day. All animals were studied on day 12 of lactation. 2.2. Circulating metabolites and tissue ATP -citrate lyase and malic enzymes activities Some animals from each group were killed by decapitation between 10:00 and 11:00 h. An aliquot of trunk blood was collected into heparinized tubes and immediately centrifuged at 4°C. Another blood aliquot was collected without anticoagulant and used for measurement of alcohol concentration by the ``headspace'' gas chromatography method (Gauvin et al., 1994) and glucose concentration by colorimetry (Dubowski, 1962). Plasma protein and triacylglycerols were determined according to the methods of Lowry et al. (1951) and Eggstein and Kreutz (1966), respectively. Samples of liver, parametrial white adipose tissue (PAR), and mammary gland (MG) were rapidly excised, placed in liquid nitrogen, and kept at ÿ70°C until analyzed for ATP citrate lyase (ATP - cit) and malic enzyme activities. For these measurements, tissues were homogenized in the extraction medium appropriate for each enzyme (1:5 w:v). The enzyme 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 of Lowry et al. (1951). 2.3. Measurement of lipogenesis rate At 10:00 h, another set of rats from each group was injected intraperitoneally with 3.0 mCi of 3H2O and killed by decapitation 1 h later. Trunk blood was collected into heparinized tubes; liver, PAR, and MG were removed; 1g aliquots were saponified in 3 ml of 30% KOH; and tissue lipid was extracted in petroleum ether (Stansbie et al., 1976). The lipogenic rate was estimated as described by Robinson et al. (1978). The fat content of the tissues was determined gravimetrically (Oller do Nascimento & Williamson, 1988).
PAR (8) (6) (11) (13)
76.6 1.24 79.38 2.99 77.69 2.74 75.33 2.37
MG (8) (5) (11) (7)
9.44 1.15 8.74 1.56 10.04 0.49 10.30 0.50
(8) (5) (9) (11)
2.4. Statistical analysis Results were expressed as mean SEM, and statistical comparison among groups was made by one -way analysis of variance. Differences between means were tested for significance by the Duncan's multiple comparison tests. Significance level was set at p< 0.05. 3. Results As shown in Fig. 1A, daily alcohol intake increased gradually throughout the experimental period in all ethanol groups. This observation suggests that a possible initial distasteful perception towards ethanol may have been gradually overcome. At day 12 of treatment, ethanol intake of E20 was higher than that of E10. Fig. 1B shows that the food intake of E5 was similar to that of the C group throughout the 12 days. On the other hand, food intake of E10 and E20 rats was significantly lower than that of C animals (25% and 60% lower, respectively). Dams of the groups E10 and E20 (Fig. 2) as well as their pups (Fig. 3) showed a significant reduction in body weight. MG and PAR relative weights were significantly reduced only in E20 (Table 1), whereas protein and lipid content of these tissues were not altered in any of the ethanol groups (Table 2). In E20, MG, and PAR, the lipogenesis rate showed a twofold increase, whereas in E5, the liver and MG lipogenesis rate had a threefold increase compared with the rates in the C group. No changes were observed in E10 (Table 3). The malic enzyme activity was decreased in the liver (E20) and MG (E10 and E20), while it was increased in the Table 3 Effect of ethanol intake by lactating rats on tissue in vivo lipogenesis rate (mmol 3H2O incorporated into lipid / g fresh tissue / h) C E5 E10 E20
Liver
PAR
MG
12.89 3.19 (8) 28.38 3.84* (11) 18.99 2.65 (13) 16.60 2.60 (5)
0.83 0.10 (7) 1.08 0.07 (6) 0.72 0.07 (10) 2.05 0.24* (5)
38.26 9.07 (7) 90.15 8.36* (10) 48.70 8.04 (11) 60.50 6.68* (6)
Values are mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
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Table 4 Effect of ethanol intake by lactating rats on tissue malic and ATP - cit enzymes activities (mmol / min / g of tissue) Malic C E5 E10 E20
ATP - cit
Liver
PAR
MG
Liver
PAR
MG
0.46 0.03 (10) 0.57 0.04* (9) 0.39 0.03 (10) 0.25 0.04* (10)
0.04 0.008 (9) 0.07 0.008 (17) 0.08 0.01 (16) 0.08 0.01 (13)
1.82 0.15 (9) 1.94 0.14 (10) 1.36 0.09* (9) 1.34 0.09* (9)
1.20 0.09 (10) 1.65 0.15* (8) 1.61 0.22* (8) 0.52 0.08* (9)
0.03 0.002 (6) 0.03 0.002 (15) 0.03 0.003 (13) 0.05 0.005* (12)
2.48 0.26 2.41 0.17 2.27 0.26 2.15 0.18
(9) (10) (10) (8)
Values are mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
liver of E5. The activity of the ATP - cit enzyme in the liver was decreased in E20 but increased in E5 and E10. This activity was increased in the PAR of E20 rats compared with the ones in the C group (Table 4). Blood glucose concentration was not altered by ethanol ingestion. However, plasma triacylglycerol concentration was higher in E10 and E20 than in the C group. Plasma protein concentration was decreased in E20 only (Table 5). Blood alcohol levels (mg / dl), measured at day 12 of lactation, were as follows: 2.87 1.06 in E5, 43.45 11.50 in E10, and 100.66 25.30 in E20. 4. Discussion The present findings showed that the metabolic changes caused by ethanol ingestion during lactation were dose - dependent. The energy required for optimal milk synthesis is high and has been shown to induce lactating rats to become hyperphagic (Williamson, 1986). In the present study, food consumption was increased during the lactation period in C, E5, and E10, although it was lower in E10 than in C rats. On the other hand, E20 rats did not develop hyperphagia. These results are in agreement with findings in a previous report (Tavares do Carmo et al., 1996). The control of eating involves neural and humoral inputs of various sorts from the periphery to the central nervous system, especially the hypothalamus. At least three neurotransmitter systems (serotonergic, opioidenergic, and dopaminegic) are strongly implicated in the control of food intake (Forsander, 1994), and alcohol
Table 5 Effect of ethanol intake by lactating rats on plasma biochemical parameters C E5 E10 E20
Glucose (mg / dl)
Protein (g / dl)
TG (mg / dl)
101.6 6.58 (8) 104.67 3.82 (5) 101.99 3.95 (11) 98.78 1.29 (5)
4.45 0.11 (7) 4.06 0.14 (6) 4.09 0.27 (5) 3.75 0.07* (5)
130.68 2.90 (7) 124.79 5.77 (5) 154.05 4.23* (10) 148.02 8.23* (5)
Values are mean SEM. Number of rates used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
ingestion reportedly altered the serotonergic system (LeMarquand et al., 1994). The blood levels of ethanol achieved in E10 and E20 were similar to those reported in previous studies (Steven et al., 1989; Tavares do Carmo et al., 1996), being twice as high in E20 than in E10. These results demonstrate a direct correlation between the ethanol concentration in the drinking water and its blood concentration. However, this correlation was not observed in E5, in which blood levels were 15 times lower than those in E10. This discrepancy could be attributed to the low food intake observed in E10 and E20, since alcohol consumption has been shown to diminish the efficiency of meal absorption (Wedel et al., 1991). Other factors, such as ethanol- induced hepatic toxicity, which leads to low ethanol metabolization by liver and ethanol neurotoxicity, which in turn leads to dehydration and hyperosmolarity due to supraoptic hypothalamic nucleus damage (Madeira et al., 1993), may also have contributed to the observed blood ethanol levels. In accordance with the alteration in food intake in E10 and E20, we observed a decrease in body weight at day 4 of lactation, which was maintained until the end of the experimental period. The body weight loss was higher in E20 (17%) than in E10 (6%). This catabolic state of E10 and E20 dams during the first four days of lactation could have been important for the pups' normal body weight gain during this period, because after that, the pups' growth was lower than that of the control group. Tavares do Carmo et al. (1996), while comparing ethanol -treated with pair-fed lactating rats, showed a similar rate of body weight decline, suggesting that this effect might be a consequence of reduced food intake (Herrera & Llobera, 1981). Despite the low food consumption in E10 and E20, glicemia levels were at the normal range. This could be accounted for by the lipolytic (Brindley, 1988) and plasma beta -hydroxybutyrate enhancing effects of ethanol (Tavares do Carmo et al., 1996). However, plasma protein concentration was low only in E20, which suggests that at the higher dosage ethanol induced undernourishment in the lactating rats. These results are in agreement with the observation that ethanol treatment decreased the hepatic synthesis and secretion of albumin (Rothschild et al., 1983). This effect has recently been shown to be dose - dependent (Volpi et al., 1998).
L.M. Oyama et al. / Alcohol 21 (2000) 195± 200
In the present study, we observed a decrease in MG and PAR relative weights in E20 rats. Although some of the effects of ethanol intake may have been a result of the possible undernourishment, others are more likely related to ethanol- intrinsic metabolism. In fact, our results showed that in E5 and E20 an increased MG lipogenesis rate occurred in the absence of food intake or body weight changes observed in E5. No concomitant increase was observed in the activities of ATP -cit and malic enzymes in this tissue. The result observed in E20 was probably not due to an increase in labeled lipid uptake by the MG. Tavares do Carmo et al. (1996) have previously observed a decrease in both lipoprotein lipase activity and accumulation of orally administered lipids in the MG of lactating rats drinking a 20% ethanol solution. Our present observation of an elevated triacylglycerols plasma concentration in E10 and E20 is consistent with this previous demonstration. Since ethanol ingestion has been shown to inhibit oxytocin release (Corio et al., 1992; Funchs, 1969), it can be proposed that in E20 a low milk release by MG could at least partially explain the increase in MG lipogenesis rate with no change in ATP -cit and malic enzyme activities. This effect was probably absent in E5 rats, as indicated by the lack of pups' growth impairment. The liver lipogenesis rate, ATP -cit, and malic enzymes activities were increased in E5. Ethanol oxidation by the liver increases the NADH /NAD + ratio (Guthrie et al., 1990), which could be an important factor favoring hepatic lipogenesis in E5 rats. Lipid production by the liver is increased during lactation to provide fatty acids for milk synthesis in the MG (Agius & Williamson, 1980). Taken together, our results suggest that in E5 the increased 3H - lipid accumulation in the MG is probably due to lipid uptake from VLDL instead of an elevation in MG lipogenesis rate. The PAR lipogenesis rate and ATP -cit enzyme activity were increased in E20. The reported ethanol- induced reduction of prolactin secretion (Subramanian, 1995) could be partially responsible for this result, since low prolactin levels have been shown to increase insulin receptors density in white adipose tissue (Vernon & Flint, 1983). The present results showed that high doses of ethanol (10% and 20% solutions) added to the drinking water induced alterations in dams' metabolism and impaired pups' growth. However, a low ethanol dose (5%) did not affect pups' body weight gain, even though it caused changes in dams' metabolism. Whether these divergent responses rely on different, dose -dependent effects of ethanol intake through breast milk on pups' metabolism remains to be elucidated. Acknowledgments The authors thank E.B. Ribeiro for reviewing the manuscript. The work was supported by CNPq and Fapesp.
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Ethanol intake during lactation I. Effects on dams' metabolism and pups' body weight gain 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 Wistar lactating rats (8 pups per dam) had free access to either tap water (control group, C) or one of three concentrations of ethanol (E) in the drinking water: 5% (E5), 10% (E10), and 20% (E20). All animals received normal rat chow ad libitum and were killed on day 12 of lactation. Intake of both 10% and 20% ethanol solutions decreased food intake, dams' body weight, and pups' body weight gain as compared with findings in the C group. The relative weights (g / 100g b.w.) of the mammary glands (MG) and of the parametrial white adipose tissue depot were decreased only in E20 as compared with findings in the C group. Protein and lipid content of these tissues were not altered in any of the ethanol groups. In comparison with the C group, the lipogenesis rate was increased in the MG (135.6%) and liver (120.2%) in E5 and the MG (58.1%) and parametrial white adipose tissue depot (147.0%) in E20. No modifications in lipogenesis rate were noted in E10. The malic enzyme activity was decreased in the MG in E10 (25.3%) and E20 (26.4%) and in the liver in E20 (45.7%). In E5, however, it was increased in the liver (23.9%). The activity of ATP - citrate lyase in the liver was decreased in E20 (56.7%), while it was increased by 37.5% in E5 and 34.2% in E10. Blood glucose concentration of dams was not affected by ethanol ingestion. However, plasma triacylglycerol concentration was higher in E10 (17.9%) and E20 (13.3%) than in the C group, and plasma protein was lower in E20 (15.7%) than in C. We concluded that alcohol intake during lactation increased the MG lipogenesis rate; although at the highest dose, this metabolic alteration was not enough to allow normal pups' growth. However, the low dose of ethanol (5%), despite having altered dams' metabolism, did not affect pups' body weight gain. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Alcohol; Lactation; Mammary gland; Liver; Lipogenesis; Rat; ATP - citrate lyase; Malic enzyme
1. Introduction Lactation is characterized by an increased demand for metabolic substrates to provide the constituents of milk (e.g., lactose, lipid, and protein). Adequate milk production and composition is highly dependent on several physiological adaptations, such as increased cardiac output; liver, heart, mammary gland, and intestine hypertrophy; and increased food intake (Chatwin et al., 1969; Craft, 1970; Fell et al., 1963).
* 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).
Chronic alcohol ingestion is associated with undernourishment due, at least in part, to a low food intake and decreased capacity for intestinal absorption (Lieber, 1984). In rats, maternal alcoholism during pregnancy resulted in severe prenatal growth deficiency (Testar et al., 1986). Vilaro et al. (1987) showed that chronic ethanol ingestion during gestation and lactation altered mammary gland function, lowering total milk production while increasing its lipid content. Accordingly, we have previously observed that the ingestion of a 20% ethanol solution by lactating rats decreased pups' body weight gain and altered the mammary gland lipid metabolism, as shown by an elevated rate of in vivo lipogenesis (Tavares - do- Carmo & Nascimento - Curi, 1990). On the other hand, mammary gland lipoprotein lipase activity and uptake of orally administered triacylglycerol were both diminished by the ethanol treatment during lactation (Tavares do Carmo et al., 1996).
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 3 - 2
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Fig. 3. Effect of ethanol intake by lactating rats on pups' body weight gain (g). The symbols are: . C; 6 E5; ~ E10; 5 E20. Values are mean SEM of 10 animals. * Significantly different from C at p < 0.05.
Fig. 1. Effect of ethanol intake by lactating rats on dams' (A) food intake (g) and (B) ethanol consumption (ml / 100 g b.w. / day). Values are mean SEM of 10 animals. * Significantly different from C at p < 0.05. + Significantly different from day 1 in the same group at p < 0.05. # Significantly different from E5 at p < 0.05.
Little et al. (1989) have demonstrated that in human beings ethanol ingested through breast milk had a slight but significant detrimental effect on motor, but not mental, development in breast - fed infants. However, a low dose of alcohol has been traditionally recommended to lactating mothers as an auxiliary factor for lactation because it
provides additional calories and fluid (Mennella & Beauchamp, 1993) and facilitates the ejection reflex (Auerbach et al., 1987). These data suggest that the safety of alcohol consumption, particularly with respect to the presence or absence of deleterious effects to lactation, may depend on the dosage consumed. However, it has been shown that low ethanol ingestion (5%) by lactating rats decreased milk retinol concentration and reduced food intake (Albuquerque et al., 1998). The aim of the present study was to examine the effect of consumption of 5%, 10%, or 20% ethanol in the drinking water during lactation on dams' lipid metabolism and its consequences to pups' body weight gain. 2. Methods The Experimental Research Committee of the Federal SaÄo Paulo University approved all procedures involving animals. 2.1. Animals and procedures Wistar lactating rats were housed in individual cages under constant conditions of lighting (12 h / 12 h) and temperature (24 1°C) and fed Nuvilab rat chow ad libitum. On the first day post partum, after adjustment of the litters to eight pups, dams were divided into four groups: a control group (C), which received drinking water ad libitum; and ethanol groups (E), which received free access to ethanol in Table 1 Effect of ethanol intake by lactating rats on tissue weight (g / 100 g)
Fig. 2. Effect of ethanol ingestion by lactating rats on dams' body weight gain (g). The symbols are: . C; 6 E5; ~ E10; 5 E20. Values are mean SEM of 10 animals. * Significantly different from C at p < 0.05. + Significantly different from day 1 in the same group at p < 0.05.
C E5 E10 E20
Liver
PAR
MG
4.11 0.09 (8) 4.30 0.16 (6) 4.09 0.13 (9) 3.95 0.14 (5)
1.57 0.19 (7) 1.44 0.22 (6) 1.55 0.08 (10) 0.80 0.21* (5)
4.58 0.27 (6) 3.96 0.15 (5) 4.27 0.22 (8) 3.21 0.12* (5)
Values are mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
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Table 2 Effect of ethanol intake by lactating rats on tissue protein and lipid content (mg / g) Protein C E5 E10 E20
Lipid
Liver
PAR
139.84 9.61 (10) 160.78 5.84 (10) 141.91 10.01 (9) 146.69 10.92 (10)
22.68 3.70 20.96 1.41 19.38 0.92 21.18 1.74
(9) (15) (14) (15)
MG
Liver
97.0 4.58 (10) 114.0 4.53 (10) 98.53 7.18 (10) 102.86 4.62 (10)
3.81 0.15 3.36 0.07 3.88 0.09 4.10 0.20
the drinking water in the concentration of 5% (E5), 10% (E10), or 20% (E20). Daily food and ethanol intakes and body weights were measured throughout the treatment period. Pups were weighed every other day. All animals were studied on day 12 of lactation. 2.2. Circulating metabolites and tissue ATP -citrate lyase and malic enzymes activities Some animals from each group were killed by decapitation between 10:00 and 11:00 h. An aliquot of trunk blood was collected into heparinized tubes and immediately centrifuged at 4°C. Another blood aliquot was collected without anticoagulant and used for measurement of alcohol concentration by the ``headspace'' gas chromatography method (Gauvin et al., 1994) and glucose concentration by colorimetry (Dubowski, 1962). Plasma protein and triacylglycerols were determined according to the methods of Lowry et al. (1951) and Eggstein and Kreutz (1966), respectively. Samples of liver, parametrial white adipose tissue (PAR), and mammary gland (MG) were rapidly excised, placed in liquid nitrogen, and kept at ÿ70°C until analyzed for ATP citrate lyase (ATP - cit) and malic enzyme activities. For these measurements, tissues were homogenized in the extraction medium appropriate for each enzyme (1:5 w:v). The enzyme 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 of Lowry et al. (1951). 2.3. Measurement of lipogenesis rate At 10:00 h, another set of rats from each group was injected intraperitoneally with 3.0 mCi of 3H2O and killed by decapitation 1 h later. Trunk blood was collected into heparinized tubes; liver, PAR, and MG were removed; 1g aliquots were saponified in 3 ml of 30% KOH; and tissue lipid was extracted in petroleum ether (Stansbie et al., 1976). The lipogenic rate was estimated as described by Robinson et al. (1978). The fat content of the tissues was determined gravimetrically (Oller do Nascimento & Williamson, 1988).
PAR (8) (6) (11) (13)
76.6 1.24 79.38 2.99 77.69 2.74 75.33 2.37
MG (8) (5) (11) (7)
9.44 1.15 8.74 1.56 10.04 0.49 10.30 0.50
(8) (5) (9) (11)
2.4. Statistical analysis Results were expressed as mean SEM, and statistical comparison among groups was made by one -way analysis of variance. Differences between means were tested for significance by the Duncan's multiple comparison tests. Significance level was set at p< 0.05. 3. Results As shown in Fig. 1A, daily alcohol intake increased gradually throughout the experimental period in all ethanol groups. This observation suggests that a possible initial distasteful perception towards ethanol may have been gradually overcome. At day 12 of treatment, ethanol intake of E20 was higher than that of E10. Fig. 1B shows that the food intake of E5 was similar to that of the C group throughout the 12 days. On the other hand, food intake of E10 and E20 rats was significantly lower than that of C animals (25% and 60% lower, respectively). Dams of the groups E10 and E20 (Fig. 2) as well as their pups (Fig. 3) showed a significant reduction in body weight. MG and PAR relative weights were significantly reduced only in E20 (Table 1), whereas protein and lipid content of these tissues were not altered in any of the ethanol groups (Table 2). In E20, MG, and PAR, the lipogenesis rate showed a twofold increase, whereas in E5, the liver and MG lipogenesis rate had a threefold increase compared with the rates in the C group. No changes were observed in E10 (Table 3). The malic enzyme activity was decreased in the liver (E20) and MG (E10 and E20), while it was increased in the Table 3 Effect of ethanol intake by lactating rats on tissue in vivo lipogenesis rate (mmol 3H2O incorporated into lipid / g fresh tissue / h) C E5 E10 E20
Liver
PAR
MG
12.89 3.19 (8) 28.38 3.84* (11) 18.99 2.65 (13) 16.60 2.60 (5)
0.83 0.10 (7) 1.08 0.07 (6) 0.72 0.07 (10) 2.05 0.24* (5)
38.26 9.07 (7) 90.15 8.36* (10) 48.70 8.04 (11) 60.50 6.68* (6)
Values are mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
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Table 4 Effect of ethanol intake by lactating rats on tissue malic and ATP - cit enzymes activities (mmol / min / g of tissue) Malic C E5 E10 E20
ATP - cit
Liver
PAR
MG
Liver
PAR
MG
0.46 0.03 (10) 0.57 0.04* (9) 0.39 0.03 (10) 0.25 0.04* (10)
0.04 0.008 (9) 0.07 0.008 (17) 0.08 0.01 (16) 0.08 0.01 (13)
1.82 0.15 (9) 1.94 0.14 (10) 1.36 0.09* (9) 1.34 0.09* (9)
1.20 0.09 (10) 1.65 0.15* (8) 1.61 0.22* (8) 0.52 0.08* (9)
0.03 0.002 (6) 0.03 0.002 (15) 0.03 0.003 (13) 0.05 0.005* (12)
2.48 0.26 2.41 0.17 2.27 0.26 2.15 0.18
(9) (10) (10) (8)
Values are mean SEM. The number of rats used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
liver of E5. The activity of the ATP - cit enzyme in the liver was decreased in E20 but increased in E5 and E10. This activity was increased in the PAR of E20 rats compared with the ones in the C group (Table 4). Blood glucose concentration was not altered by ethanol ingestion. However, plasma triacylglycerol concentration was higher in E10 and E20 than in the C group. Plasma protein concentration was decreased in E20 only (Table 5). Blood alcohol levels (mg / dl), measured at day 12 of lactation, were as follows: 2.87 1.06 in E5, 43.45 11.50 in E10, and 100.66 25.30 in E20. 4. Discussion The present findings showed that the metabolic changes caused by ethanol ingestion during lactation were dose - dependent. The energy required for optimal milk synthesis is high and has been shown to induce lactating rats to become hyperphagic (Williamson, 1986). In the present study, food consumption was increased during the lactation period in C, E5, and E10, although it was lower in E10 than in C rats. On the other hand, E20 rats did not develop hyperphagia. These results are in agreement with findings in a previous report (Tavares do Carmo et al., 1996). The control of eating involves neural and humoral inputs of various sorts from the periphery to the central nervous system, especially the hypothalamus. At least three neurotransmitter systems (serotonergic, opioidenergic, and dopaminegic) are strongly implicated in the control of food intake (Forsander, 1994), and alcohol
Table 5 Effect of ethanol intake by lactating rats on plasma biochemical parameters C E5 E10 E20
Glucose (mg / dl)
Protein (g / dl)
TG (mg / dl)
101.6 6.58 (8) 104.67 3.82 (5) 101.99 3.95 (11) 98.78 1.29 (5)
4.45 0.11 (7) 4.06 0.14 (6) 4.09 0.27 (5) 3.75 0.07* (5)
130.68 2.90 (7) 124.79 5.77 (5) 154.05 4.23* (10) 148.02 8.23* (5)
Values are mean SEM. Number of rates used in each determination is shown in parentheses. * Significantly different from C at p < 0.05.
ingestion reportedly altered the serotonergic system (LeMarquand et al., 1994). The blood levels of ethanol achieved in E10 and E20 were similar to those reported in previous studies (Steven et al., 1989; Tavares do Carmo et al., 1996), being twice as high in E20 than in E10. These results demonstrate a direct correlation between the ethanol concentration in the drinking water and its blood concentration. However, this correlation was not observed in E5, in which blood levels were 15 times lower than those in E10. This discrepancy could be attributed to the low food intake observed in E10 and E20, since alcohol consumption has been shown to diminish the efficiency of meal absorption (Wedel et al., 1991). Other factors, such as ethanol- induced hepatic toxicity, which leads to low ethanol metabolization by liver and ethanol neurotoxicity, which in turn leads to dehydration and hyperosmolarity due to supraoptic hypothalamic nucleus damage (Madeira et al., 1993), may also have contributed to the observed blood ethanol levels. In accordance with the alteration in food intake in E10 and E20, we observed a decrease in body weight at day 4 of lactation, which was maintained until the end of the experimental period. The body weight loss was higher in E20 (17%) than in E10 (6%). This catabolic state of E10 and E20 dams during the first four days of lactation could have been important for the pups' normal body weight gain during this period, because after that, the pups' growth was lower than that of the control group. Tavares do Carmo et al. (1996), while comparing ethanol -treated with pair-fed lactating rats, showed a similar rate of body weight decline, suggesting that this effect might be a consequence of reduced food intake (Herrera & Llobera, 1981). Despite the low food consumption in E10 and E20, glicemia levels were at the normal range. This could be accounted for by the lipolytic (Brindley, 1988) and plasma beta -hydroxybutyrate enhancing effects of ethanol (Tavares do Carmo et al., 1996). However, plasma protein concentration was low only in E20, which suggests that at the higher dosage ethanol induced undernourishment in the lactating rats. These results are in agreement with the observation that ethanol treatment decreased the hepatic synthesis and secretion of albumin (Rothschild et al., 1983). This effect has recently been shown to be dose - dependent (Volpi et al., 1998).
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In the present study, we observed a decrease in MG and PAR relative weights in E20 rats. Although some of the effects of ethanol intake may have been a result of the possible undernourishment, others are more likely related to ethanol- intrinsic metabolism. In fact, our results showed that in E5 and E20 an increased MG lipogenesis rate occurred in the absence of food intake or body weight changes observed in E5. No concomitant increase was observed in the activities of ATP -cit and malic enzymes in this tissue. The result observed in E20 was probably not due to an increase in labeled lipid uptake by the MG. Tavares do Carmo et al. (1996) have previously observed a decrease in both lipoprotein lipase activity and accumulation of orally administered lipids in the MG of lactating rats drinking a 20% ethanol solution. Our present observation of an elevated triacylglycerols plasma concentration in E10 and E20 is consistent with this previous demonstration. Since ethanol ingestion has been shown to inhibit oxytocin release (Corio et al., 1992; Funchs, 1969), it can be proposed that in E20 a low milk release by MG could at least partially explain the increase in MG lipogenesis rate with no change in ATP -cit and malic enzyme activities. This effect was probably absent in E5 rats, as indicated by the lack of pups' growth impairment. The liver lipogenesis rate, ATP -cit, and malic enzymes activities were increased in E5. Ethanol oxidation by the liver increases the NADH /NAD + ratio (Guthrie et al., 1990), which could be an important factor favoring hepatic lipogenesis in E5 rats. Lipid production by the liver is increased during lactation to provide fatty acids for milk synthesis in the MG (Agius & Williamson, 1980). Taken together, our results suggest that in E5 the increased 3H - lipid accumulation in the MG is probably due to lipid uptake from VLDL instead of an elevation in MG lipogenesis rate. The PAR lipogenesis rate and ATP -cit enzyme activity were increased in E20. The reported ethanol- induced reduction of prolactin secretion (Subramanian, 1995) could be partially responsible for this result, since low prolactin levels have been shown to increase insulin receptors density in white adipose tissue (Vernon & Flint, 1983). The present results showed that high doses of ethanol (10% and 20% solutions) added to the drinking water induced alterations in dams' metabolism and impaired pups' growth. However, a low ethanol dose (5%) did not affect pups' body weight gain, even though it caused changes in dams' metabolism. Whether these divergent responses rely on different, dose -dependent effects of ethanol intake through breast milk on pups' metabolism remains to be elucidated. Acknowledgments The authors thank E.B. Ribeiro for reviewing the manuscript. The work was supported by CNPq and Fapesp.
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