Hyperthyroidism Affects Lipid Metabolism in Lactating and Suckling Rats Silvia Mabel Varasa, Graciela Alma Jahnb, and María Sofía Giméneza,* a

Laboratory of Biological Chemistry, Department of Biochemistry and Biological Sciences, Faculty of Chemistry, Biochemistry, and Pharmacy, National University of San Luis, 5700 San Luis, and bLaboratory of Reproduction and Lactation, CRICYT-CONICET, 5500 Mendoza, Argentina

ABSTRACT: Two per thousand pregnant women have hyperthyroidism (HT), and although the symptoms are attenuated during pregnancy, they rebound after delivery, affecting infant development. To examine the effects of hyperthyroidism on lactation, we studied lipid metabolism in maternal mammary glands and livers of hyperthyroid rats and their pups. Thyroxine (10 µg/100 g body weight/d) or vehicle-treated rats were made pregnant 2 wk after commencement of treatment and sacrificed on days 7, 14, and 21 of lactation with the litters. Circulating triiodothyronine and tetraiodothyronine concentrations in the HT mothers were increased on all days. Hepatic esterified cholesterol (EC) and free cholesterol (FC) and triglyceride (TG) concentrations were diminished on days 14 and 21. Lipid synthesis, measured by incorporation of [3H]H2O into EC, FC, and TG, fatty acid synthase, and acetyl CoA carboxylase activities increased at day 14, while incorporation into FC and EC decreased at days 7 and 21, respectively. Mammary FC and TG concentrations were diminished at day 14; incorporation of [3H]H2O into TG decreased at days 7 and 21, and incorporation of [3H]H2O into FC increased at day 14. In the HT pups, growth rate was diminished, tetraiodothyronine concentration rose at days 7 and 14 of lactation, and triiodothyronine increased only at day 14. Liver TG concentrations increased at day 7 and fell at day 14, while FC increased at day 14 and only acetyl CoA carboxylase activity fell at day 14. Thus, hyperthyroidism changed maternal liver and mammary lipid metabolism, with decreased lipid concentration in spite of increased liver rate of synthesis and decreases in mammary synthesis. These changes, along with the mild hyperthyroidism of the litters, may have contributed to their reduced growth rate. Paper no. L8550 in Lipids 36, 801–806 (August 2001).

Two per 1000 pregnant women have hyperthyroidism (HT) (1). Most of the symptoms of hyperthyroidism are attenuated during pregnancy, but the marked rebound after delivery has impact on infant development. Specific signs in the neonate include growth retardation, advanced bone age in relation to chronological age, and craniosynostosis (2). Hyperthyroxinemia and suppressed serum thyroid-stimulating hormone (TSH) in postnatal blood confirm the diagnosis (1). Similarly, in hyperthyroid rats, TSH secretion in the pituitary is sup*To whom correspondence should be addressed at Laboratorio de Química Biológica, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Avenida Ejército de los Andes 954, 5700-San Luis, Argentina. E-mail: [email protected] Abbreviations: ACC, acetyl CoA carboxylase; Co, control rat; CPT-1, carnitine palmitoyltransferase 1; DTT, dithiothreitol; EC, esterified cholesterol; FAS, fatty acid synthase; FC, free cholesterol; HT, hyperthyroid rat = thyroxine-treated rat; LPL, lipoprotein lipase; T3, triiodothyronine; T4, tetraiodothyronine; TG, triglycerides; TSH, thyroid-stimulating hormone. Copyright © 2001 by AOCS Press

pressed, and serum and milk TSH concentrations are reduced as well (3). Lactation is associated with widespread changes in wholebody lipid metabolism, the purpose of which is to direct lipids and lipid precursors to the mammary glands for milk-fat production (4,5). Fat is the major component of the milk, with 95% of this fat being triglycerides (TG) (4). Survival of all newborn mammals is dependent upon an adequate milk supply secreted from the mammary glands of the mother (6). Triiodothyronine (T3) administration in physiological amounts is able to stimulate prolactin-induced synthesis of milk products and the enzymatic activities related to this process in differentiated rat mammary glands (7), but there is a paucity of studies concerning the effects of thyroid hormone excess on lactation and mammary gland function. Thyroid hormones affect a number of physiological processes including lipid, carbohydrate, and protein metabolism (8–10). Studies with litter-removed lactating rats have shown that hyperthyroidism depresses lipoprotein lipase (LPL) activity in mammary gland and white adipose tissue (7). Rosato et al. (11,12) showed that the chronic administration of tetraiodothyronine (T4) (100 µg/100 g body wt) produces marked changes in organ weight, lipid and protein content, and enzymatic activities (11) in virgin rats. These changes were markedly attenuated in pregnant rats near term (12) as well as in their fetuses (11), but the treatment produced advances in delivery and lactogenesis, with adverse effects on maternal behavior and milk release that resulted in death of the pups (12). The administration of 25 µg T4/100 g body wt showed similar effects on delivery and pup mortality (13). Additionally, we previously showed that chronic administration of a lower dose of 10 µg/100 g body wt of T4 in virgin females produced important changes in liver lipid metabolism (14). In the present work, we investigated the effects of chronic administration of 10 µg T4/100 g body wt on the lipid metabolism of the dams and their pups at days 7, 14, and 21 of lactation. This dose regimen was selected because it produced hyperthyroidism as measured by increases in circulating thyroid hormones and allowed the rats to nurse the litters until weaning. MATERIALS AND METHODS Chemicals and radioisotopes. [3H]H2O (3.70 GBq/g) and [14C]NaHCO3 (39.2 MBq/mmol) were purchased from Dupont, New England Company (Boston, MA). Lipid standards were acquired from Sigma Chemical Co. (St. Louis, MO). L-Tetraiodothyronine (T4) was a generous gift from Glaxo (Buenos Aires, Argentina). All the other chemicals

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were of reagent grade and were obtained from Merck Laboratory (Buenos Aires, Argentina). Animals and experimental design. Adult female Wistar rats bred in our laboratory, 3- to 4-mon-old, and weighing 190–210 g at the onset of treatment were used. The rats were housed in an animal room and kept in a 22–25°C controlled environment with a light-dark cycle of 12 h each. Rat chow and tap water were available ad libitum. Hyperthyroidism was induced by daily subcutaneous injection of T4 (at a dose of 10 µg/100 g body wt) dissolved in 0.9% NaCl and alkalinized with NaOH to pH 9. The presence of spermatozoa in the vaginal smear the morning after caging with a fertile male during the night of proestrus was indicative of pregnancy and this day was counted as day 0 of pregnancy. Thyroxine-treated (= hyperthyroid; HT) or vehicle-treated (= control; Co) rats were made pregnant approximately 14 d after commencement of treatment and sacrificed on day 7, 14, or 21 of lactation at 0900–1000 by decapitation. The rats were injected during six (L7), seven (L14), and eight (L21) weeks. On day 1 of lactation, the number of pups in each litter was standardized to eight. Trunk blood of the dams and the pups was collected, and serum was separated by centrifugation and stored at −20°C until used. The livers from dams and pups and inguinal mammary glands from the dams were removed, washed in a cold saline solution, and stored at −70°C until they were analyzed. The values are means ± standard error of the means (SEM) for groups of eight lactating rats. Blood and tissues of the pups were pooled for each litter and thus the values represent the means of the values for the litters. The body weights of the pups are the means ± SEM for eight litter means. Animal maintenance and handling were performed according to the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 86–23, revised 1985 and 1991) and the United Kingdom requirements for ethics of animal experimentation [Animals (Scientific Procedures) Act 1986]. Serum determinations. Serum T3 and T4 total concentrations were determined by a commercial enzyme-linked immunoserbant assay kit purchased from Boehringer, Mannheim, Germany. Tissue preparation and enzymatic assays. Liver portions (1 g for 4 mL of buffer) were homogenized in an Ultra Turrax T25 homogenizer (Jahnke & Kunkle, Stauffen, Germany) in 0.5 M potassium phosphate buffer (pH 7) containing 10 mM EDTA and 10 mM D,L-dithiothreitol (DTT). The homogenates were centrifuged at 100,000 × g for 1 h to yield the cytosolic fraction in a Beckman model L8-80M ultracentrifuge with a Ty-80 rotor. Cytosolic fatty acid synthase (FAS) activity was determined spectrophotometrically by a modified version of the method of Alberts et al. (15). The reaction mixture contained a 0.5 M potassium phosphate buffer (pH 6.6), 1 µmol each of EDTA and DTT, respectively, 100 nmol NADPH, and 0.05 mL of the cytosolic fraction. The reaction was started by adding 100 nmol of malonyl-CoA, and the final assay volume was 1.05 mL. The oxidation of NADPH at 30°C was monitored at 340 nm. Acetyl CoA carboxylase (ACC) activity in liver or mammary cytosols was measured as described previously by Allred and Rochringer (16). The enzyme activity was measured using Lipids, Vol. 36, no. 8 (2001)

a reaction mixture that, in a final volume of 700 µL, contained 60 mM buffer Tris-acetate (pH 7.8), 100 mM potassium acetate, 3 mM DTT, 8.5 mM potassium citrate, 1 mM ATP, 0.6 mg/mL bovine serum albumin, 0.35 mM acetyl CoA, 8 mM magnesium acetate, 25 mM sodium bicarbonate, and 2 µCi [14C]NaHCO3. For blanks, acetyl-CoA was omitted. The mixture was preincubated 1 min at 37°C. Then 50 µL of the cytosolic fraction was added. After 1 min of incubation at 37°C, 50 µL of concentrated HCl was added to stop the reaction. A 200 µL aliquot was transferred into the scintillation vial and dried under cold air flow. The dried extract was resuspended with 200 µL 50% ethanol, and the radioactivity was measured in 10 mL of scintillation fluid in a Wallac LKB 1409 liquid scintillation analyzer. ACC activity was expressed as units per mg of protein, where 1 unit equals 1 pmol of [14C]bicarbonate incorporated into malonyl-CoA per minute at 37°C. The protein concentration was determined by the method of Lowry et al. (17), using fraction V bovine serum albumin as standard. Lipid determinations. The lipids from the hepatic or mammary tissue were extracted with chloroform/methanol (2:1) according to the method of Folch et al. (18). An aliquot of the lipid extracts was taken to determine total cholesterol, and another one to separate the different lipid fractions by thin-layer chromatography with an n-hexane/diethyl ether/acetic acid (80:20:1, by vol) solvent system. After eluting the scraped bands, aliquots were used for the mass determination according to the methods of Sardesai and Manning (19) for TG and of Zack et al. (20) after saponification (21) for free cholesterol (FC) and esterified cholesterol (EC). A recovery from thin-layer chromatography averaging 90% of cholesterol mass was obtained. Incorporation of 3H from H2O into lipids. The groups of lactating rats were fed ad libitum and then injected intraperitoneally with 3H2O (3.7 mBq/rat in 9 g/L of NaCl). They were killed 1 h later to ensure that the newly synthesized lipids in the liver and mammary gland had been labeled. One gram of the liver and mammary gland was extracted with 20 mL of chloroform/methanol (2:1) according to the method of Folch et al. (18). The radioactivity incorporated into the lipids was counted in the different lipid fractions that had been separated by thin-layer chromatography (see section above on lipid determinations). The results are expressed as pg 3H incorporated/h/g of tissue. Statistical analyses. Significant differences among means were considered at a level of P < 0.05 and identified by oneway analysis of variance and the Tukey test. In all cases, the variances were homogeneous. RESULTS Serum T4 and T3 concentrations in Co and HT lactating rats and their pups. Serum T4 and T3 concentrations in the HT group of lactating rats were increased significantly compared with the Co group (Fig. 1). The serum T4 concentrations of the pups of the HT lactating dams were also increased significantly compared with the offspring of Co lactating dams on days 7 and 14 of neonatal

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life, but not on day 21. The serum T3 concentrations increased significantly on day 14 compared with the pups of the Co lactating dams, but no differences were observed on days 7 and 21 (Fig. 1). Body, liver weight, and total protein of liver and mammary gland in HT and Co lactating rats and their pups. Table 1 shows that the liver weight of the HT mothers was increased significantly compared with Co only on day 7 of lactation. No differences were observed in body weight or in liver or mammary gland protein concentrations in any of the other studied days. The body weights of the pups of the HT lactating dams were decreased significantly compared with the Co on all the days of lactation. The total protein concentrations and liver weights on day 7 of lactation were lower than in the Co group, but no differences were observed on days 14 and 21 of lactation. TG, cholesterol concentrations, and activities of FAS and ACC in livers of the pups of HT and Co lactating rats. The liver TG concentrations in the HT group were significantly decreased on days 14 and 21 of lactation when compared with the Co group, but no significant differences were observed on day 7 (Table 2). FC and EC concentrations were also significantly lower in the HT group on days 14 and 21, but no changes in cholesterol concentrations were observed on day 7 (Table 2). As shown in Table 2, the FAS and ACC liver activities in the HT groups were higher than those of the Co on day 14 of lactation, while no differences were observed on the other days of lactation.

FIG. 1. Effects of hyperthyroidism on plasma thyroxine (T4) and triiodothyronine (T3) in lactating rats and their pups. Rats were made hyperthyroid (HT) by daily injections of T4 at a dose of 10 µg/100 g body weight and mated 2 wk after beginning the treatments. Controls (Co) were injected with vehicle. The adult rats and their litters were sacrificed on days 7, 14, and 21 of lactation. T4 and T3 were measured by enzyme-linked immunosorbent assay. Values are means ± standard error of the means for groups of eight dams or eight litters. Asterisk (*) indicates P < 0.05 compared with the respective control groups using one-way analysis of variance and the Tukey test.

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Incorporation of 3H2O into liver of HT and Co lactating rats. The incorporation of [3H] from 3H2O into liver TG was significantly increased on day 14 of lactation compared to Co. On the other hand, incorporation into FC was significantly decreased on day 7, while incorporation into both FC and EC was increased on day 14. The incorporation in the EC was lower on day 21 compared with the Co group (Table 2). TG, cholesterol concentrations, and activities of FAS and ACC in livers of the pups of HT and Co lactating rats. The TG concentrations in the HT group were increased significantly on day 7 and decreased on day 14 of lactation when compared with the Co group, but no differences were observed on day 21. FC concentrations in the pups of both HT lactating and Co dams were lower on day 14 compared with the other days of lactation. On this day the HT group had slightly higher values compared with the Co group, while there were no differences on days 7 and 21. On the other hand, there were no differences in the EC concentration between HT and Co groups on all the studied days, although an increase was observed in both groups on day 21 compared with the previous days (Table 2). FAS liver activities decreased on day 7; however, there were no differences between HT and Co pups on days 14 and 21. The ACC liver activities decreased on day 14 but no differences were observed on days 7 and 21 of lactation (Table 2). Incorporation of 3H2O into lipids in liver of the pups of HT and Co lactating rats. As shown in Table 2, no significant differences between HT and Co groups were observed in the incorporation of [3H] from 3H2O into liver TG, FC or EC fractions. On the other hand, 3H2O incorporation into both fractions of cholesterol was increased on day 14 compared with day 7 and thereafter decreased to very low values on day 21 of lactation. Mammary gland TG and cholesterol concentrations and the incorporation of 3H from 3H2O into the lipids of dams rats. The mammary TG concentrations of the HT group showed lower values on day 14 compared with the Co group, while no changes were observed on days 7 and 21 of lactation. The concentration of TG was elevated on day 14 in both groups compared with the other days. FC concentrations were also decreased only on day 14 in the HT group when compared to Co, while no changes in EC concentrations were observed in the 3 d of lactation studied (Table 3). In the mammary gland, there was a decrease in the incorporation of [3H] into TG in the HT groups on days 7 and 21 of lactation compared with the Co, with no differences on day 14 of lactation. There was an increase in incorporation into FC in the HT group only on day 14 of lactation compared with the Co, while no differences were observed in the incorporation into EC fractions in the three studied days (Table 3). DISCUSSION The present paper describes the changes in lipid metabolism caused by chronic T4 treatment during the days previous to pregnancy, during pregnancy, and during lactation in the mammary gland and liver in the dams on days 7, 14, and 21 of lactation and the effects on the pups. Lipids, Vol. 36, no. 8 (2001)

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TABLE 1 Effects of Hyperthyroidism on Body and Liver Weight and Liver Total Protein Concentration in Lactating Rats and Their Pupsa Day 7 of lactation Co HT Mothers Liver weight (g) Total liver protein (mg/g) Total mammary protein (mg/g) Body weight (g) Pups Liver weight (g) Total liver protein (mg/g) Body weight (g)

Day 14 of lactation Co HT

Day 21 of lactation Co HT

9.2 ± 0.5 104.2 ± 15.1 121.6 ± 11.1 250.7 ± 5.8

11.2 ± 0.2b 109.3 ± 16.4 103.4 ± 6.8 251.1 ± 11.1

10.7 ± 1.1 187.4 ± 0.4 128.0 ± 8.1 253.0 ± 7.2

12.0 ± 0.3 186.2 ± 2.9 119.8 ± 8.8 257.8 ± 5.2

11.5 ± 0.4 181.9 ± 28.1 122.5 ± 4.3 260.2 ± 12.5

10.8 ± 0.9 137.9 ± 16.9 111.7 ± 3.5 258.7 ± 2.3

0.49 ± 0.03 146.2 ± 5.91 14.71 ± 0.70

0.40 ± 0.01b 110.5 ± 7.41b 10.42 ± 1.01b

0.88 ± 0.04 168.00 ± 7.57 25.74 ± 0.67

0.84 ± 0.04 158.37 ± 7.34 19.14 ± 1.81b

1.46 ± 0.17 167.4 ± 10.23 39.60 ± 1.74

1.65 ± 0.11 149.9 ± 8.64 24.74 ± 2.95b

a Values are means ± standard error of the means for groups of eight dams or eight litters. Co, control—vehicle-treated rats; HT, hyperthyroid—thyroxinetreated rats. b P < 0.05 compared with the respective control groups using one-way analysis of variance and the Tukey test.

TABLE 2 Effects of Hyperthyroidism on Liver Triglyceride and Cholesterol Concentrations and the Incorporation of 3H from 3H2O into the Lipids and Lipogenic Enzyme Activities of Lactating Rats and Their Pupsa Day 7 of lactation Co HT Mothers Triglyceride (TG) (µg/g of liver) Free cholesterol (FC) (µg/g of liver) Esterified cholesterol (EC) (µg/g of liver) FAS ACC 3 H Incorporation To TG (ng 3H/h/g of liver) To FC(ng 3H/h/g of liver) To EC (ng 3H/h/g of liver) Pups Triglyceride (µg/g of liver) FC (µg/g of liver) EC (µg/g of liver) FAS ACC 3 H Incorporation To TG (ng 3H/h/g of liver) To FC (ng 3H/h/g of liver) To EC (ng 3H/h/g of liver)

1630 ± 115 2700 ± 109 515 ± 47 0.49 ± 0.03 76.83 ± 1.91

2909 ± 686 2561 ± 173 464 ± 27 0.62 ± 0.17 87.65 ± 5.36

Day 14 of lactation Co HT

Day 21 of lactation Co HT

3551 ± 377 6473 ± 485 6268 ± 1221 1.64 ± 0.11 83.87 ± 4.65

1027 ± 128b 5158 ± 305b 3370 ± 253b 3.30 ± 0.36b 115.73 ± 5.82b

2305 ± 244 3527 ± 229 2775 ± 239 6.18 ± 0.75 105.62 ± 15.1

1423 ± 275b 1960 ± 218b 1362 ± 296b 6.19 ± 0.32 128.8 ± 9.9

6.86 ± 0.73 6.11 ± 0.76 4.04 ± 1.26

5.44 ± 0.63 3.00 ± 0.59b 3.63 ± 0.78

3.51 ± 0.43 4.05 ± 0.32 2.35 ± 0.61

5.38 ± 0.23b 5.17 ± 0.34b 4.16 ± 0.32b

3.90 ± 0.44 1.72 ± 0.24 2.06 ± 0.09

3.90 ± 0.65 1.57 ± 0.31 0.82 ± 0.17b

855 ± 132 1802 ± 336 471 ± 109 0.93 ± 0.21 16.1 ± 0.7

1239 ± 65b 2551 ± 621 560 ± 90 0.24 ± 0.05b 17.5 ± 1.9

2356 ± 227 333 ± 34 401 ± 45 1.68 ± 0.12 19.9 ± 1.3

1627 ± 132b 619 ± 57b 440 ± 67 1.75 ± 0.4 14.2 ± 0.6b

1243 ± 177 2638 ± 334 1231 ± 106 1.12 ± 0.27 29.3 ± 5.9

1053 ± 147 3057 ± 184 1193 ± 146 1.21 ± 0.26 27.7 ± 3.3

1.48 ± 0.33 1.40 ± 0.23 0.67 ± 0.07

3.24 ± 0.82 1.60 ± 0.40 1.08 ± 0.18

2.57 ± 0.43 2.88 ± 0.38 2.53 ± 0.73

3.66 ± 0.32 3.20 ± 0.37 2.73 ± 0.35

3.81 ± 0.20 0.30 ± 0.01 0.23 ± 0.02

3.62 ± 0.52 0.40 ± 0.11 0.26 ± 0.01

a

Values are means ± standard error of the means for groups of eight dams or eight litters. Fatty acid synthase (FAS) and acetyl coenzyme A carboxylase (ACC) activities were expressed as units/mg of cytosolic proteins; for other abbreviations see Table 1. b P < 0.05 compared with the respective control groups using one-way analysis of variance and the Tukey test.

TABLE 3 Effects of Hyperthyroidism on Mammary Gland Triglyceride and Cholesterol Concentrations and the Incorporation of 3H from 3H2O into the Lipids of Lactating Ratsa Day 7 of lactation Co HT TG (µg/g of MG) FC (µg/g of MG) EC (µg/g of MG) Incorporation To TG (ng 3H/h/g of MG) To FC (ng 3H/h/g of MG) To EC (ng 3H/h/g of MG) a

Day 14 of lactation Co HT

Day 21 of lactation Co HT

3.18 ± 0.22 2042 ± 330 849 ± 221

3.62 ± 0.43 1724 ± 290 1346 ± 326

40.37 ± 3.15 1661 ± 73 503 ± 82

22.32 ± 3.90b 1153 ± 119b 515 ± 38

2.10 ± 0.17 3531 ± 353 915 ± 86

2.30 ± 0.20 3286 ± 204 964 ± 105

32.94 ± 1.37 1.41 ± 0.24 1.09 ± 0.13

28.05 ± 0.75b 1.42 ± 0.12 1.12 ± 0.03

28.20 ± 2.89 3.03 ± 0.11 2.38 ± 0.13

29.36 ± 6.0 3.98 ± 0.40b 2.60 ± 0.43

28.66 ± 8.74 1.60 ± 0.26 1.90 ± 0.94

13.34 ± 3.72b 1.31 ± 0.09 0.98 ± 0.05

Values are means ± standard error of the means for groups of eight dams. MG, mammary gland; for other abbreviations see Table 1. P < 0.05 compared with the respective control groups using one-way analysis of variance and the Tukey test. For abbreviations see Table 2.

b

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Effects of hyperthyroidism in lactating dams. The dose of 10 µg of T4 per 100 g body wt induced an increase in the circulating concentration of T3 and T4 in the dams at the three times of lactation studied, thus confirming the state of HT. Long-chain fatty acids are structural components of all cells; they also serve as energy reserves and are involved in a variety of regulatory functions including their own synthesis and degradation (22). ACC is the enzyme that catalyzes the rate-limiting step in the biogenesis of long-chain fatty acids. It is well known that thyroid hormones increase FAS and ACC activities at a pretranslational step (23). In our experimental model the activities of FAS and ACC in the livers of HT rats increased on day 14 of lactation, corresponding to the increased synthesis of TG as observed by the 3H2O incorporation experiments, and accompanied by a paradoxical decrease in the mass of liver TG. Similar results were observed in HT virgin females (11), but no changes were found in enzyme activities on days 7 and 21 of lactation in spite of the decreased content of TG on day 21. Increased malonyl-CoA, which is only generated by ACC, suppresses mitochondrial carnitine palmitoyltransferase I activity (CPT-I) (24); this may increase the level of cytosolic longchain fatty acyl-CoA esters, which are known signals for insulin secretion (25). But, in the hyperthyroid state, fatty acid oxidation and ketogenesis are stimulated simultaneously along with a paradoxical stimulation of fatty acid synthesis (26). These processes may be linked, because thyroid hormones accelerate fatty acyl-CoA entry into the mitochondrial matrix by increasing mitochondrial CPT-I activity (27) and decreasing sensitivity of CPT-I to inhibition by malonyl-CoA (26). The liver plays a central role in the maintenance of whole-body cholesterol by integrating the regulation of a group of hepatic enzymes, receptors, and other proteins important for cholesterol homeostasis. During lactation the synthesis of cholesterol is increased, in part to provide de novo synthesized cholesterol for new membrane synthesis in the liver hypertrophy that occurs during lactation and also for lipoprotein secretion to provide cholesterol and triglycerides for milk production (28). Sixty percent of the total content of cholesterol in milk is of hepatic origin (29). The lower content of FC in the livers of the HT mothers on day 14 of lactation compared to the control group, associated with the increased synthesis, may indicate that export from the liver to the mammary glands is also increased. On the other hand, in thyroxine-treated rats, an increase in the activity of hepatic acyl coenzyme A:cholesterol acyl transferase (ACAT) has been reported (30). Consistent with this, we also observed an increase in the synthesis of EC on day 14 of lactation in the HT rat compared with the Co. The higher activities of hepatic lipogenic enzymes in the hyperthyroid rats suggest that there are sufficient fatty acids available for cholesterol esterification. However, the EC mass decreased in the HT rats, suggesting that export of EC from liver to mammary gland was also augmented. In mammary tissue, acidic cholesterol ester hydrolase activities increase during lactation and fall 2 d after weaning, increasing the FC concentration destined to be secreted into milk (31). In our experimental model, we observed no differences in EC content and rate of synthesis in the HT rat compared with Co on

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day 7 or 21; however, on day 14 both groups showed the lowest EC content. On the other hand, in the HT group, FC concentration on day 14 of lactation was diminished, in spite of an increase in its synthesis, suggesting that there is an increased mobilization of cholesterol from the gland to milk on this day. Milk TG and cholesterol are known to be derived from lipoproteins as well as originated from mammary de novo synthesis (28). On day 7 we observed a decrease in the synthesis of TG in the HT groups, without changes in TG tissue content. However, on day 14 there was a decrease in the mass of TG compared with the high values observed in the Co, while no changes were observed in the synthesis, which decreased on day 21. It is well known that milk stasis induces involution of the tissue (32). The decrease in TG synthesis and content observed on day 21, along with the reduced growth rate of the litter and histological changes (not shown), are suggestive of a premature mammary involution in the HT mothers. Our results show that the most pronounced effects of HT in liver and mammary gland lipid metabolism were on day 14 of lactation, with effects that were similar to those observed previously in the virgin females. Day 14 may be considered the peak of lactation, when the growth rate of the litter is maximal, and, thus, the demand on maternal metabolism, especially mammary metabolism, may be at its greatest and the effects of HT most marked. In contrast, on day 7 no changes were observed, and on day 21 of lactation there was an amelioration of the effects observed on day 14. Moreover, the changes observed on day 21 could be associated with premature weaning and involution of the mammary glands. Effects of maternal HT on the metabolism of the pups. We observed an increase in T4 concentration in the HT pups at 7 and 14 d but not at 21 d old, suggesting that there is an important contribution of thyroid hormone from the milk. However, the T3 concentration only increased in the pups at 14 d of life. Our results agree with previous data showing that the conversion rates of T4 to T3 in livers of pups before day 10 are very low and then rise to reach the maximal value at around day 23 of life (33). The T3 and T4 concentrations in pups at 21 d of life were not modified, probably because on this day the pups were already eating solid food and drinking water. Chronic administration of T4 to lactating rats negatively affected the growth rate of the litters in the 3 d studied, as is shown by the decrease in their body weights. These results agree with Rosato et al. (11), who showed that the administration of T4 (100 µg/100 g body wt) produced an advance in lactogenesis in the mammary gland associated with a 100% mortality in the pups, suggesting deficient milk release, as the pups were unable to draw milk from the mothers in spite of vigorous suckling. Additionally we observed that the birth weights of the pups of hyperthyroid rats were smaller than the control group (HT: 6.11 ± 0.008 g and Co: 6.67 ± 0.11 g; P < 0.001). Moreover, we found that the liver weight in 7-d-old pups was also diminished. This decrease may be related to a loss in liver glycogen (HT: 47.15 ± 5.35 and Co: 110.86 ± 37.26 µM glucose/g of liver; P < 0.05) associated with a reduction in protein content. Although thyroid hormones have been reported to stimulate the expression of ACC and FAS mRNA in the liver of the adult Lipids, Vol. 36, no. 8 (2001)

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rat, some evidence suggests that they do not play a primary role in the increase of lipogenic enzyme mRNA concentration after weaning to a high-carbohydrate, low-fat diet (34). Additionally, studies performed in suckling rats injected with T3, during 2 d, reported no changes in the activity of the liver FAS (35). We observed in the liver of the pups a decrease in FAS activity at day 7 and of ACC activity at day 14 of lactation. On day 7 of lactation the liver TG of the pups are medium-chain TG and longchain TG proceeding from the milk. The increased TG content at day 7 may be linked to the fact that thyroid hormone accelerates the absorption of lipids into the mucosal cells of the intestinal tract (36). Maternal hyperthyroidism produces complex changes in liver and mammary lipid metabolism, which may be partially responsible for the diminished growth of the litter in combination with the hyperthyroid state of the pups themselves. ACKNOWLEDGMENTS This work has been supported by grants PIP 0826; PIP 4931, from CONICET (National Investigation Council of Science and Technology, Argentina); PICT 09130 from FONCYT (Argentine Agency for the Promotion of Science); and by Project 8104 from San Luis University, Argentina.

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