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• W2013513900 abstract "We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk.The observed changes indicate an active role of HSL in mammary gland lipid metabolism. We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism. Mammary glands, besides being among the tissues in the body with the highest lipid content after adipose tissue (1Lopez-Luna P. Maier I. Herrera E. Carcass and tissue fat content in the pregnant rat.Biol. Neonate. 1991; 60: 29-38Crossref PubMed Scopus (57) Google Scholar), are one of the most active metabolic tissues in the body during pregnancy and lactation. Milk lipid is an important source of both calories and essential fatty acids for the newborn. During lactation, women secrete 800 ml of milk per day containing 4% fat, mostly corresponding to triacylglycerols, of which the mammary gland synthesizes ∼32 g daily (2Allen J.C. Keller R.P. Archer P.C. Neville M.C. Studies in human lactation. VI. Milk composition and daily secretion rates of macronutrients in the first year lactation.Am. J. Clin. Nutr. 1991; 54: 69-80Crossref PubMed Scopus (176) Google Scholar). The lactating mouse mammary gland secretes 5 ml of milk per day containing ∼30% fat (3Schwertfeger K.L. McManaman J.L. Palmer C.A. Neville M.C. Anderson S.M.M. Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation.J. Lipid Res. 2003; 44: 1100-1112Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). To develop this transitory capacity for handling such a large amount of lipids, the morphology of the mammary tissue changes during pregnancy and lactation. In nonpregnant mammary glands, the predominant cells are adipocytes with epithelial structures interdispersed among them. During pregnancy, in mammary glands there is an extensive proliferation of alveolar structures into the adipocytes, accompanied by differentiation of epithelial cells (4Robinson G.W. McKnight R.A. Smith G.H. Hennighausen L. Mammary epithelial cells undergo secretory differentiation in cycling virgins but require pregnancy for the establishment of terminal differentiation.Development. 1995; 121: 2079-2090Crossref PubMed Google Scholar), which show cytoplasmic lipid droplets (5Neville M.C. Physiology of lactation.Clin. Perinatol. 1999; 26: 251-279Abstract Full Text PDF PubMed Google Scholar) surrounded by the protein adipophilin (6McManaman J.L. Palmer C.A. Wright R.M. Neville M.C. Functional regulation of xanthine oxidoreductase expression and localization in the mouse mammary gland: evidence of a role in lipid secretions.J. Physiol. (Lond.). 2002; 545: 567-579Crossref Scopus (110) Google Scholar). During lactation, epithelial cells are the predominant cell type, and only small channels of lipid-filled adipocytes and lipid-depleted adipocytes may be distinguished. During pregnancy, major changes in maternal lipid metabolism occur. Fat depots accumulate during the early stages of pregnancy and decrease during the late phases (7Herrera E. Lasunción M.A. Gomez-Coronado D. Aranda P. Luna P. Lopez Maier I.I. Role of lipoprotein lipase activity on lipoprotein metabolism and the fate of circulating triglycerides in pregnancy.Am. J. Obstet. Gynecol. 1988; 158: 1575-1583Abstract Full Text PDF PubMed Scopus (161) Google Scholar). Net catabolic changes taking place in adipose tissue during late pregnancy are manifested by an enhanced hormone-sensitive lipase (HSL) activity and decreased LPL activity (8Martin-Hidalgo A. Holm C. Belfrage P. Schotz M.C. Herrera E. Lipoprotein lipase and hormone-sensitive lipase activity and mRNA in rat adipose tissue during pregnancy.Am. J. Physiol. 1994; 266: E930-E935PubMed Google Scholar), which result in an increase in maternal plasma lipids both in humans (9Konttinen A.T. Pyorala T. Carpen E. Serum lipid pattern in normal pregnancy and preeclampsia.J. Obstet. Gynaecol. Br. Commonw. 1964; 71: 453-458Crossref PubMed Scopus (25) Google Scholar, 10Knopp R.H. Montes A. Warth M.R. Carbohydrate and lipid metabolism in normal pregnancy.in: National Research Council Committee on Nutrition of the Mother and Preschool Child Laboratory Indices of Nutritional Status in Pregnancy. National Academy of Sciences, Washington, DC1978: 35-88Google Scholar) and in rats (11Scow R.O. Chernick S.S. Brinley M.S. Hyperlipemia and ketosis in the pregnant rat.Am. J. Physiol. 1964; 206: 796-804Crossref PubMed Scopus (84) Google Scholar, 12Knopp R.H. Sandek C.D. Arky R.A. O'Sullivan J.B. Two phases of adipose tissue metabolism in pregnancy: maternal adaptations for fetal growth.Endocrinology. 1973; 92: 984-988Crossref PubMed Scopus (113) Google Scholar). The increments of triglyceride (TG)-rich lipoproteins (chylomicrons and VLDLs) are among the most pronounced changes in plasma lipids during late pregnancy (10Knopp R.H. Montes A. Warth M.R. Carbohydrate and lipid metabolism in normal pregnancy.in: National Research Council Committee on Nutrition of the Mother and Preschool Child Laboratory Indices of Nutritional Status in Pregnancy. National Academy of Sciences, Washington, DC1978: 35-88Google Scholar, 13Alvarez J.J. Montelongo A. Iglesias A. Lasunción M.A. Herrera E.E. Longitudinal study on lipoprotein profile, high density lipoprotein subclass, and postheparin lipases during gestation in women.J. Lipid Res. 1996; 37: 299-308Abstract Full Text PDF PubMed Google Scholar, 14Argiles J. Herrera E. Lipids and lipoproteins in maternal and fetus plasma in the rat.Biol. Neonate. 1981; 39: 37-44Crossref PubMed Scopus (49) Google Scholar). Reduced adipose tissue LPL activity during late gestation allows blood TG to be diverted from storage in adipose tissue to other tissues, such as mammary glands, where there is an induction in LPL expression and activity, allowing the subsequent hydrolysis and uptake of circulating TG in preparation for lactation (15Hamosh M. Clary T.R. Chernick S.S. Scow R.O. Lipoprotein lipase activity of adipose and mammary tissue and plasma triglyceride in pregnant and lactating rats.Biochim. Biophys. Acta. 1970; 210: 473-482Crossref PubMed Scopus (200) Google Scholar, 16Ramirez I. Llobera M. Herrera E. Circulating triacylglycerols, lipoproteins, and tissue lipoprotein lipase activities in rat mothers and offspring during the perinatal period: effect of postmaturity.Metabolism. 1983; 32: 333-341Abstract Full Text PDF PubMed Scopus (64) Google Scholar). Besides the enhanced LPL-dependent uptake of TG from plasma TG-rich lipoproteins (17Williamson D.H. Integration of metabolism in tissues of the lactating rat.FEBS Lett. 1980; 117S: K93-K105Crossref Scopus (98) Google Scholar, 18Cryer A. Tissue lipoprotein lipase activity and its action in lipoprotein metabolism.Int. J. Biochem. 1981; 13: 525-541Crossref PubMed Scopus (280) Google Scholar), during lactation mammary epithelial cells synthesize TG from fatty acids obtained from the blood stream and from their de novo synthesis from glucose (19Neville M.C. Picciano M.F. Regulation of milk lipid secretion and composition.Annu. Rev. Nutr. 1997; 17: 159-184Crossref PubMed Scopus (221) Google Scholar). The cytoplasmic lipid droplets formed move toward the apical surface of the epithelial cells and are budded into the lumen. This process results in secretion of the milk lipid globules that are surrounded by the membrane, composed of both apical plasma membrane and intracellular components (20Mather I.H. Keenan T.W. Origin and secretion of milk lipids.J. Mammary Gland Biol. Neoplasia. 1998; 3: 259-273Crossref PubMed Scopus (287) Google Scholar). HSL, the key enzyme in lipolysis, is an intracellular neutral lipase that hydrolyzes triacylglycerols, diacylglycerols, monoacylglycerols, and cholesteryl and retinyl esters (21Yeaman S.J. Hormone-sensitive lipase—new roles for an old enzyme.Biochem. J. 2004; 379: 11-22Crossref PubMed Scopus (198) Google Scholar, 22Fredrickson G. Stralfors P. Nilsson N. Ö. Belfrage P. Hormone-sensitive lipase of the rat adipose tissue: purification and some properties.J. Biol. Chem. 1992; 256: 6311-6320Abstract Full Text PDF Google Scholar, 23Cook K.G. Yeaman S.J. Stralfors P. Fredrikson G. Belfrage P. Direct evidence that cholesterol ester hydrolase from adrenal cortex is the same enzyme as hormone-sensitive lipase from adipose tissue.Eur. J. Biochem. 1982; 125: 245-249Crossref PubMed Scopus (100) Google Scholar, 24Wei S. Lai K. Patel S. Piantedosi R. Shen H. Colantuoni V. Kraemer F.B. Blaner W.S.S. Retinyl ester hydrolysis and retinol efflux from BFC-1β adipocytes.J. Biol. Chem. 1997; 272: 14159-14165Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 25Fredrickson G. Tornqvist H. Belfrage P. Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of adipocyte triacylglycerol.Biochim. Biophys. Acta. 1986; 876: 288-293Crossref PubMed Scopus (156) Google Scholar). The activity of HSL is regulated posttranslationally by phosphorylation-dephosphorylation reactions. The activation by fast-acting lipolytic hormones (catecholamines, isoproterenol, glucagon, adrenocorticotropic hormone) involves a hormone receptor-induced increase in the intracellular concentration of cAMP, which activates cAMP-dependent protein kinase A. Protein kinase A then phosphorylates HSL, resulting in an increase in hydrolytic activity (26Stralfors P. Belfrage P. Phosphorylation of hormone-sensitive lipase by cyclic AMP-dependent protein kinase.J. Biol. Chem. 1983; 258: 15146-15152Abstract Full Text PDF PubMed Google Scholar). Dephosphorylation is affected primarily by protein phosphatases, which are activated by insulin (27Stralfors P. Björgell P. Belfrage P. Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin.Proc. Natl. Acad. Sci. USA. 1984; 81: 3317-3321Crossref PubMed Scopus (198) Google Scholar). Although HSL expression is highest in adipose tissue, HSL is also expressed in brown adipose tissue, adrenals, corpus luteum, testis, ovary, and, to a lesser extent, skeletal and cardiac muscle and macrophages (23Cook K.G. Yeaman S.J. Stralfors P. Fredrikson G. Belfrage P. Direct evidence that cholesterol ester hydrolase from adrenal cortex is the same enzyme as hormone-sensitive lipase from adipose tissue.Eur. J. Biochem. 1982; 125: 245-249Crossref PubMed Scopus (100) Google Scholar, 28Holm C. Belfrage P. Fredrickson G. Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue.Biochem. Biophys. Res. Commun. 1987; 148: 99-105Crossref PubMed Scopus (132) Google Scholar, 29Cook K.G. Colbran R.J. Snee J. Yeaman S.J. Cytosolic cholesterol ester hydrolase from bovine corpus luteum. Its purification, identification, and relationship to hormone-sensitive lipase.Biochim. Biophys. Acta. 1983; 752: 46-53Crossref PubMed Scopus (52) Google Scholar, 30Stenson Holst L. Hoffmann A.M. Mulder H. Sundler F. Holm C.C. Localization of hormone-sensitive lipase to rat Sertoli cells and its expression in developing and degenerating testes.FEBS Lett. 1994; 355: 125-130Crossref PubMed Scopus (34) Google Scholar, 31Kraemer F.B. Patel S. Saedi M.S. Sztalryd C. Detection of hormone-sensitive lipase in various tissues. I. Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies.J. Lipid Res. 1993; 34: 663-671Abstract Full Text PDF PubMed Google Scholar). The HSL gene is located on chromosome 19q13.3 (32Holm C. Kirchgessner T.G. Svenson K.L. Fredrickson G. Nilsson S. Miller C.G. Shively J.E. Heinzmann C. Sparkes R.S. Mohandas T. Lusis A.J. Belfrage P. Schotz M.C.C. Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.Science. 1988; 241: 1503-1506Crossref PubMed Scopus (266) Google Scholar) and was initially described as containing nine exons, which encode the adipocyte protein, spanning 11 and 10 kb in humans (33Langin D. Laurell H. Holst L.S. Belfrage P. Holm C. Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an Antarctic bacterium.Proc. Natl. Acad. Sci. USA. 1993; 90: 4897-4901Crossref PubMed Scopus (160) Google Scholar) and mice (34Li Z. Sumida M. Birchbauer A. Schotz M.C. Reue K. Isolation and characterization of the gene for mouse hormone-sensitive lipase.Genomics. 1994; 24: 259-265Crossref PubMed Scopus (42) Google Scholar), respectively. Interestingly, the size of HSL mRNAs is variable. Rat heart, skeletal muscle, placenta, and ovaries express slightly larger HSL mRNAs (3.5 kb) than adipose tissue (3.3 kb) (32Holm C. Kirchgessner T.G. Svenson K.L. Fredrickson G. Nilsson S. Miller C.G. Shively J.E. Heinzmann C. Sparkes R.S. Mohandas T. Lusis A.J. Belfrage P. Schotz M.C.C. Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.Science. 1988; 241: 1503-1506Crossref PubMed Scopus (266) Google Scholar). The testis is characterized by expression of an even larger mRNA species of 3.9 kb (32Holm C. Kirchgessner T.G. Svenson K.L. Fredrickson G. Nilsson S. Miller C.G. Shively J.E. Heinzmann C. Sparkes R.S. Mohandas T. Lusis A.J. Belfrage P. Schotz M.C.C. Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.Science. 1988; 241: 1503-1506Crossref PubMed Scopus (266) Google Scholar). The purified adipose tissue enzyme has a molecular mass of 84 and 88 kDa in rats (32Holm C. Kirchgessner T.G. Svenson K.L. Fredrickson G. Nilsson S. Miller C.G. Shively J.E. Heinzmann C. Sparkes R.S. Mohandas T. Lusis A.J. Belfrage P. Schotz M.C.C. Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.Science. 1988; 241: 1503-1506Crossref PubMed Scopus (266) Google Scholar) and humans (33Langin D. Laurell H. Holst L.S. Belfrage P. Holm C. Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an Antarctic bacterium.Proc. Natl. Acad. Sci. USA. 1993; 90: 4897-4901Crossref PubMed Scopus (160) Google Scholar), respectively. The testis appears to express two isoforms, one 84 kDa protein that is similar to adipose HSL, and a second larger isoform of ∼120–130 kDa, encoded by a single 3.9 kb HSL mRNA in rat testis (30Stenson Holst L. Hoffmann A.M. Mulder H. Sundler F. Holm C.C. Localization of hormone-sensitive lipase to rat Sertoli cells and its expression in developing and degenerating testes.FEBS Lett. 1994; 355: 125-130Crossref PubMed Scopus (34) Google Scholar) and by both 3.3 and 3.9 kb HSL mRNAs in human testis (35Stenson Holst L. Langin D. Mulder H. Laurell H. Grober J. Bergh A. Mohrenweiser W. Edgren G. Holm C.C. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase.Genomics. 1996; 35: 441-447Crossref PubMed Scopus (83) Google Scholar, 36Mairal A. Malanie N. Laurell H. Grober J. Holst L. Stenson Guillaudeux T. Holm C. Jégou B. Langin D.D. Characterization of a novel testicular form of human hormone-sensitive lipase.Biochem. Biophys. Res. Commun. 2002; 291: 286-290Crossref PubMed Scopus (32) Google Scholar). Although we and others have previously described the presence of HSL activity in mammary glands of lactating rats (37Tavares do Carmo M.G. Oller do Nascimento C.M. Martín-Hidalgo A. Herrera E.E. Effects of ethanol intake on lipid metabolism in the lactating rat.Alcohol. 1996; 13: 443-448Crossref PubMed Scopus (14) Google Scholar, 38Small C.A. Yeaman S.J. West D.W. Clegg R.A. Cholesterol ester hydrolysis and hormone-sensitive lipase in lactating rat mammary tissue.Biochim. Biophys. Acta. 1991; 1082: 251-254Crossref PubMed Scopus (15) Google Scholar) and human mammary gland contains HSL mRNA (35Stenson Holst L. Langin D. Mulder H. Laurell H. Grober J. Bergh A. Mohrenweiser W. Edgren G. Holm C.C. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase.Genomics. 1996; 35: 441-447Crossref PubMed Scopus (83) Google Scholar), the role of HSL in mammary gland metabolism is largely unknown. To attain a better understanding of the potential function of HSL in the mammary gland, the present work was addressed at determining the cell types that might express this protein and the changes that take place in its expression and activity in the mammary gland during pregnancy and lactation in the rat. In addition, because insulin is well known as the most active antilipolytic hormone (39Belfrage P. Fredrikson G. Olsson H. Stralfors P. Molecular mechanisms for hormonal control of adipose tissue lipolysis.Int. J. Obes. 1985; 9: 129-135PubMed Google Scholar, 40Holm C. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.Biochem. Soc. Trans. 2003; 31: 1120-1124Crossref PubMed Scopus (0) Google Scholar) and during late pregnancy and lactation the mammary gland remains highly sensitive to it (41Burnol A-F. Loizeau M. Girard J. Insulin receptor activity and insulin sensitivity in mammary gland of lactating rats.Am. J. Physiol. 1990; 259: E828-E834Crossref PubMed Google Scholar, 42Carrascosa J.M. Ramos P. Molero J.C. Herrera E. Changes in the kinase activity of the insulin receptor account for an increased insulin sensitivity of mammary gland in late pregnancy.Endocrinology. 1998; 139: 520-526Crossref PubMed Scopus (17) Google Scholar), the study was extended to determine the potential relationship between plasma insulin levels and mammary gland HSL activity during those same stages. Female Sprague-Dawley rats from our colony, weighing 180–200 g, were maintained at 22–24°C under standard conditions of illumination (from 8:00 AM to 8:00 PM) and feeding (Purina Chow diet; Panlab, Barcelona, Spain). The experimental protocol was approved by the Animal Research Committee of the Hospital Ramón y Cajal, Madrid, Spain. The experimental groups were age- and sex-matched virgin, pregnant (days 12, 20, and 21), and lactating (days 1 and 4) rats. At least six rats were analyzed per time point. The rats were mated with normal males, and positive pregnancy was determined by the appearance of spermatozoids in vaginal smears. Litter sizes were adjusted to 9–11 pups at birth. Rats were killed between 10:00 and 11:00 AM by decapitation, and blood from the neck wound was collected in heparinized tubes. Plasma was immediately separated after blood collection by centrifugation at 4°C and kept at −80°C until analysis for immunoreactive insulin, using an ELISA kit specific for rats (Mercodia AB). The left fourth (inguinal) mammary glands and the lumbar adipose tissue were removed, snap frozen in liquid nitrogen, and stored at −80°C until processing. The right inguinal mammary glands were removed and fixed in 4% paraformaldehyde in PBS for 6–24 h at 4°C. The tissues were embedded in paraffin using conventional methods (43González-Santander R. Cuadrado G.M. Martínez M.G.S. Alonso J. Lobo M.V.V. The use of different fixatives and hydrophilic embedding media (Historesin™ and Unicryl™) for the study of embryonic tissues.Microsc. Res. Tech. 1997; 36: 1-8Crossref PubMed Scopus (97) Google Scholar) and sectioned at 5 μm, either to be stained with hematoxylin and eosin or to be used for immunohistochemistry. To determine the presence of immunoreactive HSL in milk, milk samples were collected from 15 day postpartum lactating rats (n = 5). The mothers were separated from the litters for a period of 4 h (9:00 AM to 1:00 PM) before milking. Milk samples were collected after an intraperitoneal injection of oxytocin (10 IU/rat) by manual expression of the teats while the rats were under pentobarbital anesthesia (35 mg/kg body weight). Milk samples were immediately frozen at −20°C until analyzed. Milk samples were thawed (37°C) and shaken continuously during pipetting to ensure sample uniformity. Whole milk was sonicated (three bursts, 5 s duration per burst, maximum power) and diluted in an assay buffer before assay. To remove milk lipids that would interfere in the HSL assay, milk samples were centrifuged at 1,500 g for 20 min at 4°C, and infranatants were ultracentrifuged at 110,000 g at 4°C for 45 min and used for the assay. Total HSL activity was measured as previously described (44Tornqvist H. Björgell P. Krabish L. Belfrage P. Monoacylmonoalkylglycerol as a substrate for diacylglycerol hydrolase activity in adipose tissue.J. Lipid Res. 1978; 19: 654-656Abstract Full Text PDF PubMed Google Scholar). Briefly, tissue samples were homogenized in 3 volumes of 0.25 M sucrose containing 1 mM EDTA, 1 mM dithioerythreitol, and 10 μg/ml antipain, pH 7.4. Infranatants were obtained by centrifugation at 110,000 g in a Beckman centrifuge (model TL-100) for 45 min at 4°C. A phospholipid-stabilized emulsion of a dioleoylglycerol ether analog, 1(3)-mono[3H]oleyl-2-O-oleoylglycerol, was used to assay the hormone-sensitive diacylglycerol lipase activity (36Mairal A. Malanie N. Laurell H. Grober J. Holst L. Stenson Guillaudeux T. Holm C. Jégou B. Langin D.D. Characterization of a novel testicular form of human hormone-sensitive lipase.Biochem. Biophys. Res. Commun. 2002; 291: 286-290Crossref PubMed Scopus (32) Google Scholar). Inhibition experiments were performed by preincubation with 100 mM NaF. Total cellular RNA was extracted from frozen rat mammary glands by use of a single-step acid guanidinium thiocyanate-phenol-chloroform extraction method (45Chomczynski P. Sacchi N. Single step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar) (Ultraspect RNA; Biotech). Briefly, tissues were homogenized with a Polytron in the presence of homogenization buffer. RNA was purified via a series of ethanol precipitations and quantified by optical density at 260 nm. Equal amounts (10 μg) of total RNA were denatured and fractionated on 1% agarose gels containing 2.2 M formaldehyde. Electrophoresis was carried out for 18 h at 50 V in 3-(N-morpholino)propanesulfonic acid, pH 7.0, running buffer. RNA was transferred to a nylon membrane (Hybond N+; Amersham) for 1 h in 3 M NaCl, 0.3 M sodium citrate, pH 7.0, by a capillary system and immobilized by cross-linking with ultraviolet light (46Church G.M. Gilbert W. Genomic sequencing.Proc. Natl. Acad. Sci. USA. 1984; 81: 1991-1995Crossref PubMed Scopus (7267) Google Scholar). The nylon membranes were prehybridized for 1 h at 60°C in 0.5 M sodium phosphate, pH 7.0, 1 mM EDTA, 7% (w/v) SDS, and 1% (w/v) BSA. Northern hybridization was performed with denatured 32P-labeled cDNA probes (1 × 106 cpm/ml) for 17–18 h at 60°C in the same buffer. Full-length rat HSL cDNA probe (generously provided by Dr. Cecilia Holm, University of Lund, Sweden) was radiolabeled as described by Feinberg and Vogelstein (47Feinberg A.P. Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16653) Google Scholar) using an oligolabeling kit (LKB Biotechnology, Pharmacia). DNA (25–50 ng) was labeled to a specific activity of 1–2 × 106 dpm/μg using [32P]deoxycytidine triphosphate (3,000 Ci/mmol; Amersham). Northern blot filters were washed twice (20 min each wash) with 0.3 M NaCl, 30 mM sodium citrate, pH 7.0, 0.1% SDS at room temperature and twice (20 min each wash) with 15 mM NaCl, 1.5 mM sodium citrate, pH 7.0, 0.1% SDS at 60°C. Autoradiography was performed with a single intensifying screen at −80°C and quantified by densitometric scanning. Northern blot analysis of whole mammary gland RNA was done for each animal. There were no significant differences in loading, as verified by ethidium bromide staining of the gels. Bands corresponding to 28S rRNA were quantified from the photographs of the gels, and these values were used as an internal standard. Tissues were homogenized in a buffer containing 0.25 M sucrose, 1 mM EDTA, 1 mM dithioerythreitol, 20 μg/ml antipain, and 20 μg/ml leupeptin, pH 7.4. Cellular proteins were dissolved in SDS-PAGE sample solution [60 mM Tris, pH 6.5, 10% (w/v) glycerol, 5% (w/v) β-mercaptoethanol, 20% (w/v) SDS, and 0.025% (w/v) bromphenol blue] by boiling (2 × 5 min), sonication (2 × 5 min in a sonication bath), and vortex mixing (2 × 30 s). The total amount of protein loaded in each line was always 10 μg. After fractionation by SDS-PAGE on slab gels (7 × 14 cm), proteins were electroblotted onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) using a blotting apparatus (Bio-Rad Laboratories, Hercules, CA). Reference proteins were myosin [molecular weight (Mr) 209,000], β-galactosidase (Mr 137,000), BSA (Mr 84,000), carbonic anhydrase (Mr 44,000), soybean trypsin inhibitor (Mr 32,800), lysozyme (Mr 18,700), and aprotinin (Mr 7,200) (all from Bio-Rad). Blots were blocked for 2 h in 10% defatted dry milk-TBS-0.1% Tween and then incubated for 2 h at room temperature with primary antibody (1:20,000) (chicken polyclonal antiserum directed against HSL from white rat adipose tissue; generously provided by Dr. Cecilia Holm). This was then followed by a 1 h incubation with secondary peroxidase-conjugated antibody (1:20,000) [donkey anti-chicken IgY (IgG)(H+l); RDI Research Diagnostics, Inc.] in 5% defatted dry milk-TBS-0.1% Tween. All steps were performed at room temperature, and blots were rinsed between incubation steps with TBS-0.1% Tween. Immunoreactive bands were visualized using the enhanced chemiluminescence detection method according to the manufacturer's instructions (Amersham, Dübendorf, Switzerland) and subsequent exposure of the membrane to X-ray film. Protein determination was performed using the Bradford dye method (48Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216357) Google Scholar), with Bio-Rad reagent and BSA as the standards. Mammary gland paraffin sections (5 μm thick) from rats were mounted at the indicated times on silanized slides and allowed to dry overnight before immunohistochemical staining. Paraffin was removed with xylene. Sections were hydrated, and endogenous peroxidase activity was inhibited by incubation with 3% H2O2 for 10 min and 0.3% H2O2 in methanol for an additional 20 min. Sections were then washed in TBS and incubated in 3% normal goat serum, 0.01% Triton X-100, and 0.1% glycine in TBS, pH 7.6, at room temperature for 30 min to prevent nonspecific binding of the first antibody. Afterward, the sections were incubated for 12 h at 4°C with the primary antibody, anti-HSL (chicken polyclonal antiserum directed against HSL from white rat adipose tissue; 1:100). The sections were washed twice in TBS to remove unbound primary antibody and then incubated with the secondary antibody for 1 h at room temperature. The biotinylated secondary antibody used was goat anti-chicken IgY (1:200) for HSL (Vector Laboratories, Inc., Burlingame, CA). Sections were washed in TBS and incubated with the streptavidin-peroxidase complex (Zymed Laboratories, Inc., San Francisco, CA) for 30 min and then washed in TBS followed by Tris-HCl buffer, pH 7.6. The peroxidase activity was revealed using 3-diaminobenzidine tetrahydrochloride as chromogen (Sigma, St. Louis, MO). The sections were counterstained by Carazzi's hematoxylin. Thereafter, the sections were dehydrated in ethanol, mounted in DePeX (Serva), and observed with a light" @default.
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• W2013513900 date "2005-04-01" @default.
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• W2013513900 title "Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation" @default.
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