Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation

Abstract

Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65–70% by volume compared to 25–30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling.Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism. Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65–70% by volume compared to 25–30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism. Milk lipid is an important source of both calories and essential fatty acids for the newborn. The unique demands of lactation make the lactating mammary gland among the most active triglyceride-synthesizing organs in the body. For example, a fully lactating woman secretes 800 ml of milk containing 4% fat per day; thus, the mammary gland synthesizes about 32 g of triglyceride daily or nearly 6 kg fat during a typical 6 month lactation (1Allen J.C. Keller R.P. Archer P.C. Neville M.C. Studies in human lactation: 6. Milk composition and daily secretion rates of macronutrients in the first year of lactation.Am. J. Clin. Nutr. 1991; 54: 69-80Google Scholar). The mammary gland of the lactating mouse performs an even more prodigious feat: in the FVB strain used in the experiments described in these studies, the mammary glands are estimated to secrete about 5 ml of milk per day containing about 30% lipid, 1 or 1.5 g of lipid per day. Over the course of 20 days of lactation, the dam thus secretes about 30 g of milk lipid, equivalent to her entire body weight! When one considers that the capacity for synthesizing this amount of lipid must be developed during a 20 day pregnancy and activated precisely at the time of birth, the regulatory mechanisms in both the developmental (pregnancy) and secretory (lactation) phases become extremely interesting. Mammary gland development during pregnancy is characterized by extensive proliferation of alveolar structures accompanied by differentiation of mammary epithelial cells that occurs progressively throughout pregnancy and can be monitored temporally by the regulated expression patterns of specific genes (2Robinson 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: 2079-2090Google Scholar, 3Gardner H.P. Belka G.K. Wertheim G.B.W. Hartman J.L. Ha S.I. Gimotty P.A. Marquis S.T. Chodosh L.A. Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland.Development. 2000; 127: 4493-4509Google Scholar, 4Chodosh L. Gardner H. Rajan J. Stairs D. Marquis S. Leder P. Protein kinase expression during murine mammary development.Dev. Biol. 2001; 219: 259-276Google Scholar). For example, increased expression of β-casein is observed early in pregnancy, whereas expression of whey acidic protein (WAP) occurs during mid-pregnancy (2Robinson 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: 2079-2090Google Scholar). In addition, cytoplasmic lipid droplets can be observed in the epithelial cells of the developing alveoli during mid and late pregnancy (5Neville M.C. Physiology of lactation.Clin. Perinatol. 1999; 26: 251-279Google Scholar, 6Liu X. Robinson G. Hennighausen L. Activation of Stat5a and Stat5b by tyrosine phosphorylation is tightly linked to mammary gland differentiation.Mol. by the protein J.L. Neville M.C. regulation of expression and in the mouse mammary of a role in lipid Scholar). the mammary gland milk in to both a decrease in the of by the of the and the high of by the (5Neville M.C. Physiology of lactation.Clin. Perinatol. 1999; 26: 251-279Google Scholar). lactation, mammary epithelial cells fatty acids both from the and by from glucose M.C. of milk lipid secretion and Nutr. Scholar). The cytoplasmic lipid droplets the of the epithelial cells and are into the results in secretion of the milk lipid by a of both and and secretion of milk Mammary Biol. Scholar). The in the milk lipid and of encoding an in mammary and in with the during Biol. Scholar, S. to the milk fat specific in of lactating mammary epithelial Biol. G. of in epithelium and and protein is into milk a of milk lipid J. Scholar). is that glucose by mammary epithelial cells for during fatty of is by the glucose glucose Expression of during lactation, and a in observed in mammary epithelial cells during lactation of the glucose in lactating mouse mammary 2000; Scholar, J. expression in mammary J. Scholar, S. and of glucose transport in and from lactating mammary J. Scholar). protein kinase is a protein kinase that is activated in to a of that Akt in to a of growth from cell and S. J. The an Scholar, and Akt protein kinase are and sufficient for the of growth Scholar, J. of of kinase and of of epithelial Biol. Scholar, kinase cell by the of from the Biol. 1999; Scholar, P. of by and Scholar, S. S. growth cells for Akt Biol. 1999; Scholar, a in 1999; Scholar). In to role in the of Akt is now to be in lipid and glucose in For example, expression of constitutively activated Akt in resulted in differentiation into associated with increased glucose and lipid accumulation in the of R.A. Expression of a constitutively active Akt kinase in glucose and glucose Biol. Scholar, P. D. J. Expression of a constitutively activated of protein kinase in cells Scholar). in that Akt expression of the glucose transport R.A. Expression of a constitutively active Akt kinase in glucose and glucose Biol. Scholar, S.T. J. J. R.A. of by the kinase Biol. 1999; Scholar, Protein the of but or in by a Biol. 1999; Scholar, J. H. of and glucose in to of of Protein and in Biol. 1999; Scholar). Akt is activated in to a of and growth that are in mammary gland growth S. S. growth cells for Akt Biol. 1999; growth P. P. of of protein kinase by and J. Scholar, H. R.A. of by the protein kinase growth J. S. J. Activation of growth an for a role of Akt in this 1999; G. an essential and expression during mammary gland 2001; Scholar, J. J. R.A. S. J. The differentiation is essential for mammary gland 2000; L. P. role for protein kinase in the of in 2000; and is for and protein kinase Biol. suggesting that Akt a in mammary gland development and this transgenic mice that a constitutively active of Akt the of the terminal of the mouse mammary tumor Mammary gland is by activated Akt in transgenic 2001; Scholar). We that myr-Akt expression results in with a delay in the of with the of results by used transgenic Akt J. J. J. Activation of Akt kinase in mammary epithelium a cell for tumor Biol. 2001; Scholar, S. S. mammary gland in transgenic Scholar). of mammary glands from the myr-Akt transgenic mice that Akt to regulation of milk secretion by the accumulation of cytoplasmic lipid droplets in mammary epithelial The of the transgenic mice described Mammary gland is by activated Akt in transgenic 2001; Scholar). FVB mice were from were by the of the of by the and Mammary gland were from both wild-type and transgenic mice during pregnancy and lactation. The mammary glands were in for and protein and at The mammary glands were and in The were in and at 5 to be with and or to be used for For the pregnancy mice were and daily for the of a The day of the observed was and were at or FVB and transgenic mice were per For the lactation mice were and the were to pups The day of was and were at and FVB and transgenic mice were per of the pups in these they were with of the from In the pups were daily to weight the pups in the were pups were in the weight they were nursed by the was from of were and containing and in to The were in and 50% and containing at for the were in which of containing of at The were in for at by in for at for the were and to the were by and The Akt was from The was from the of this were in with from at mice per Protein was from by in 1 5 and a The were for and Protein were the protein of protein were to and with a of the were to Mammary glands were from mice at the in and in were at 5 with was for 20 in The were with in with in and with the Mammary gland from and of pregnancy were with an protein and at a of Mammary gland from and 9 of lactation were with an from of at a of The were with at a at a and were a for with a and and The and volume of lipid droplets by were by of the by of the lipid droplets were the of to 30 mice were to mice at of and the were to pups of lactation, the mice were to the mice were with units of to milk The milk was from the mammary glands For of fat milk was into and at for 20 The amount of fat that into the was a of the in the For milk protein protein was the protein of protein were a The was with for in and for at Milk were and from a of wild-type and transgenic mice. from Neville of were in a of and containing 5 and were from the which was from were described S. R.P. J. of kinase 2000; Scholar). For cells were with the of of adenovirus for For the studies, the cells were in for 1 The cells were with of for The cells were with with and was from by in containing by at for 6 and experiments were in the cells were described and in growth for The cells were in and for 1 in The cells were with and with for described J.L. of and by with Scholar). The cells were with and to The was to and at the expression of Akt throughout mammary gland development in the wild-type of Akt The was first with a for with a and of Akt expression were to The decrease in expression observed during lactation observed in experiments and of by the of the milk protein or result from a of during lactation The used in these with and the results in the of Expression of Akt was during pregnancy, and but increased of pregnancy and of lactation high was 9 of lactation By in and Mammary gland is by activated Akt in transgenic 2001; Akt was in mammary epithelial cells during lactation. an important role for Akt during lactation. the of Akt in mammary gland transgenic mice that an activated of in mammary epithelial cells the of the Mammary gland is by activated Akt in transgenic 2001; Scholar). The expression of the myr-Akt was characterized in mammary glands from transgenic mice throughout mammary gland development by an the myr-Akt protein are observed Mammary gland is by activated Akt in transgenic 2001; Scholar). Expression of the myr-Akt increased during pregnancy both the of the and an increase in the of epithelial cells during was a in the amount of myr-Akt protein at the of lactation, by the for a time to the at this time The in expression of myr-Akt late pregnancy and lactation is more than expression by and the of in this line these with or an of the myr-Akt is constitutively active We that the of by with the with the of phosphorylation of a for Akt Mammary gland is by activated Akt in transgenic 2001; Scholar). the expression of myr-Akt results in a during pregnancy, mammary glands from both wild-type and transgenic mice throughout and of mammary glands from wild-type and and transgenic and mice during and late of the mammary glands were used to the of alveolar development and and were used to alveoli and of pregnancy, alveolar development is in wild-type and transgenic mice were at at this and development appeared at of pregnancy mammary glands from wild-type and transgenic mice and at a that cytoplasmic lipid droplets were in the epithelial cells at mid pregnancy in both the cytoplasmic lipid droplets were significantly larger in the transgenic than in the wild-type mice. of pregnancy, the of alveoli and was in the transgenic mice compared with the wild-type mice even at the cytoplasmic lipid droplets were larger in the transgenic mice, and the to the of these lipid droplets cytoplasmic lipid accumulation in mammary epithelial cells of myr-Akt transgenic mice during pregnancy, of mammary glands at and of pregnancy an is in the protein of cytoplasmic and lipid droplets J.L. Neville M.C. regulation of expression and in the mouse mammary of a role in lipid Scholar, is a specific of lipid accumulation in cell and Scholar). in was observed in from wild-type mice at of pregnancy In was observed in the alveoli from transgenic mice at this of cytoplasmic lipid volume the increase in lipid accumulation in transgenic mice of pregnancy, was in alveoli from both wild-type and transgenic mice an increase in both the and volume of cytoplasmic lipid droplets in mammary epithelial cells from transgenic mice compared with from wild-type mice during pregnancy the the and and that expression of myr-Akt in mammary epithelial cells results in precocious lipid accumulation early in pregnancy, an increase in the and of cytoplasmic lipid droplets during late myr-Akt expression results in a lactation mammary gland was during lactation and from both wild-type and transgenic mice of lactation, the alveoli to the fat in mammary glands from both wild-type and transgenic mice, and the are In addition, cytoplasmic lipid droplets can be observed in the epithelial cells they are than observed during pregnancy of lactation in mammary glands from transgenic mice, the alveoli to be and the cytoplasmic lipid droplets to be larger than observed in the wild-type mice 9 of lactation in mammary glands from wild-type mice, the alveoli the of the fat and lipid droplets are observed in the to the epithelial cells In the alveoli in mammary glands from transgenic mice and compared with alveoli from wild-type mice In addition, the lipid droplets observed in the alveoli are larger in and can be the of the in the of from the epithelial cells in the In addition, to be more that a in the alveolar compared with wild-type alveoli a result of milk a in transgenic with lactation H. and J. L. mice recombinant human protein in lactation and mammary gland In Scholar). at of lactation, the larger lipid droplets and in mammary glands from transgenic mice compared with from wild-type mice and cytoplasmic lipid in mammary glands from myr-Akt transgenic mice, were the of cytoplasmic lipid droplets during lactation that the increased in mammary glands from transgenic mice compared with from wild-type mice these cytoplasmic lipid volume and were and to be increased in the transgenic mice compared with wild-type mice that during lactation, the increase in the volume of the lipid droplets is more than the increase in the of lipid droplets. with the that expression of myr-Akt in mammary epithelial cells is lipid with the during birth, the pups from the transgenic mothers appeared compared with from wild-type and wild-type growth was a amount of was observed in the of pups nursed by the transgenic mothers and one day of they appeared than pups from wild-type When the of growth was by for 9 days a decrease in the weight was observed compared with the of pups nursed by wild-type mice, in that the in weight was a of the the both wild-type and transgenic and the pups litter were the weight at and during lactation, that the of the was a both wild-type and transgenic mothers were observed to and suggesting that are to in development in are with a lactation defect in myr-Akt transgenic mice. the of the lactation was an mammary gland from both wild-type and transgenic mice at and 9 of lactation expression is by which the of the epithelial is by and are by protein expression in mammary gland from wild-type mice was observed at the of the epithelial cells during lactation In expression can be throughout the which is to the of milk that in mammary gland from transgenic mice, expression is throughout the during lactation that the milk is from the mammary results are with the in the and that the mammary glands from the myr-Akt transgenic mice are milk during lactation. We the that milk are normally in the of the mammary glands from mice and that this protein is during the of the for this were the the of in the of mammary glands from transgenic mice the that the composition of the milk by the transgenic mice is and that in these is with a the lactation defect observed in lactating myr-Akt milk was from wild-type and transgenic mice and for both the of milk fat milk protein the milk was in and the amount of fat was and the of volume of in the Milk from wild-type Akt mice milk fat milk from transgenic mice 65–70% milk fat The protein composition of milk from wild-type and transgenic mice was of milk protein were a and with to the in the composition of the milk appeared to be in milk from wild-type and transgenic mice, be that the protein of milk from transgenic mice was significantly than in milk from wild-type mice that expression of myr-Akt in the mammary gland results in increased milk with defect in milk protein myr-Akt is increased milk lipid that lipid in a of the mammary this the of myr-Akt expression glucose and lipid accumulation in cells L. Neville M.C. of active transport mammary and of Scholar, of the glucose to of CIT3 mouse mammary epithelial Biol. 2001; which were from the mouse mammary epithelial cell J. D. mouse mammary cell line in and differentiation in Scholar). expression of myr-Akt in the of the cells to was in the expression of myr-Akt at an of resulted in increased of Expression of at an of that this is specific for myr-Akt than an of We the of myr-Akt to the of cytoplasmic lipid droplets in the a cells with myr-Akt or were with which J.L. of and by with Scholar). extensive the was from the cells and the amount of was by the at J.L. of and by with Scholar). in expression of myr-Akt resulted in increased at an of compared with both and results that myr-Akt expression can both the of and the of cytoplasmic lipid droplets in mammary epithelial results that are with the observed in the myr-Akt transgenic mice. results that of an activated of Akt in the mammary glands of transgenic mice increased lipid in both pregnancy and lactation and is associated with a lactation defect that The of the observed is to the expression of the by of this in a of the mammary epithelium to an increase in both lipid and glucose suggesting that the increase in lipid be a of increased glucose in the of the development during pregnancy is regulated by the and S. of mammary gland development and in the Scholar, S. Smith but in mice with a of the J. Scholar, J. D. H. P.A. of the in the Scholar, G. 1995; and by growth in the growth in mice a that results in 1995; Scholar, of the and genes for in mouse mammary gland 1999; and growth D. J. G. growth a role in development of the mammary Scholar). growth and to of Akt S. S. growth cells for Akt Biol. 1999; Scholar, P. P. of of protein kinase by and J. Scholar, L. P. role for protein kinase in the of in 2000; Scholar, G. by the growth and Biol. Scholar, X. P. growth and is for body 2000; Scholar). at the of these that expression of activated Akt in mammary epithelial cells alveolar of the mammary glands from the myr-Akt transgenic mice in the growth of the alveolar is that expression of the in the mammary gland during pregnancy is high to increase alveolar or that plays role in this of mammary differentiation during pregnancy is the of cytoplasmic lipid droplets at about mid droplets in with of the pregnancy and are from the into the of the mammary alveoli during pregnancy and lactation an increase in both the and of cytoplasmic lipid droplets during We observed an increase in the of these milk fat during lactation in the transgenic mice. these by with an specific to which is a protein that is in the protein of both cytoplasmic lipid droplets and the milk fat protein is into milk a of milk lipid J. Scholar). of this to lipid and in wild-type and transgenic mice and that lipid and cytoplasmic lipid is in the transgenic mice. Although these that Akt a role in or is important to that expression of Akt is late pregnancy and lactation L. Gardner H. Rajan J. Stairs D. Marquis S. Leder P. Protein kinase expression during murine mammary development.Dev. Biol. 2001; 219: 259-276Google the mammary epithelial cells are that of activated Akt are to about differentiation lipid or that Akt be a regulatory during In is that Akt is in the epithelium of the mammary gland in to a of lipid is important to that expression of Akt during pregnancy was at the that the phosphorylation and of Akt is during In to the observed in the myr-Akt transgenic mice that described Mammary gland is by activated Akt in transgenic 2001; these mice a defect in lactation. lactation defect is characterized by an decrease in the weight of pups over the first 9 days birth, and the of a of pups over the first 5 days was in the of the pups from of transgenic mothers that could for the in of transgenic and mouse that a lactation defect that is the result of development of the alveolar structures during the mice J. D. H. P.A. of the in the mice X. Robinson G. L. Hennighausen L. is for mammary gland development and mice J. J. R.A. S. J. The differentiation is essential for mammary gland 2000; and mice D. J. D. Neville is a of secretory differentiation and but in the mouse mammary gland.Development. Scholar). In development of the alveolar structures to normally in the myr-Akt transgenic mice, suggesting that is for the lactation For this milk was and from both wild-type and transgenic mice during lactation. Milk from wild-type mice of milk whereas milk from the transgenic mice 65–70% milk is important to that the milk from the transgenic mice was and a amount of milk could be compared with wild-type mice the of milk fat could be milk that could be that the of milk fat in the milk that was could be even a for the lactation the milk be that the pups be to remove the milk from the mammary to milk is that of and could to the lactation defect In this be that in a in the amount of β-casein or in lactating mice, and in these were in the mammary glands from transgenic mice than in mammary glands from mice Mammary gland is by activated Akt in transgenic 2001; Scholar). the of β-casein or is a of the of be that the of is that the of are is to a in the mammary gland expression of in the The described results were observed in line line to of the myr-Akt by and the lactation the of observed during is diminished Mammary gland is by activated Akt in transgenic 2001; Scholar). We observed changes in the of lipid droplets in that of the of the described in line be in line characterized these mice. The accumulation of the that milk is during lactation in the transgenic mice that milk can the of characterized by of the secretory epithelial cells J. H. phases of in mammary gland and Scholar). For example, milk by or results in accumulation of milk and lipid in the alveolar and epithelial cell and alveolar X. Robinson G. Hennighausen L. P.A. cell during the first of mammary gland Scholar). Although the alveoli from transgenic mice milk they to be involution. the myr-Akt is We that expression of the myr-Akt that the myr-Akt even milk first occurs and the epithelium from the pups are to at of the was to in mice, which L. D. Hennighausen L. of mammary gland and of the secretory epithelium in the of Scholar). mice could lactation to 6 days compared with days in the wild-type Akt to glucose and lipid in P. D. J. Expression of a constitutively activated of protein kinase in cells Scholar, S.T. J. J. R.A. of by the kinase Biol. 1999; Scholar, L.A. of in of glucose and Biol. 1999; Scholar). the results from the myr-Akt transgenic mice, the of myr-Akt to glucose and lipid in mammary epithelial expression of myr-Akt in the mouse mammary epithelial cell resulted in both increased of and of lipid droplets. Although the of Akt of glucose is fully that Akt glucose Akt both to the and of R.A. Expression of a constitutively active Akt kinase in glucose and glucose Biol. Scholar, S.T. J. J. R.A. of by the kinase Biol. 1999; Scholar, Protein the of but or in by a Biol. 1999; Scholar, The glucose Scholar). glucose are in the mammary of the glucose in lactating mouse mammary 2000; Scholar, J. expression in mammary J. Scholar, S. and of glucose transport in and from lactating mammary J. S. Expression and of and glucose in the and lactating mammary and most expression throughout late pregnancy and during lactation, the for glucose is high of the glucose in lactating mouse mammary 2000; Scholar). is that myr-Akt the expression or of glucose transport in mammary epithelial In this is to that the of by the with or in with glucose transport in the mammary and that with this H. S. D. regulation of the glucose in the lactating mammary Scholar). is to a increase in in mammary gland S. of the glucose in and a increase in in of expression in the mammary Scholar). to lipid in mammary of lipid in mouse mammary gland Scholar). the glucose transport and triglyceride in the mammary and the that Akt is more of fatty P. in the regulation of and genes by and a role for the regulatory J. The results that expression of myr-Akt in mammary glands of transgenic mice mammary epithelial cell differentiation by precocious development of cytoplasmic lipid droplets during In addition, the results described that myr-Akt both the of by milk lipid during lactation. and in a of the mammary epithelium the that Akt in the mammary of of and regulation of glucose in lipid The to the of the Mammary for and this was by from the to and and was by a from the glucose mouse mammary tumor whey acidic protein