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Breastfeeding offers a broad spectrum of health benefits for infants. However, overnutrition and a steady increase in maternal obesity in the U.S. have made it harder for many mothers to produce and express breastmilk, and the quality of milk from obese mothers is also frequently compromised. Adipocytes, the primary cell type in the non-lactating breast, display a drastic morphological and functional change during lactation in mice. Lipid-filled adipocytes undergo lipolysis, and lipid droplets disappear to provide fatty acids and energy for breastmilk production. Once the animal stops lactation, these lipid-depleted adipocytes return as lipid-laden cells. This dynamic remodeling of the tissue is likely the result of active intercellular communications. Connexin43 (Cx43) is the most abundant connexin in the mammary adipose tissue that makes up the gap junctions for direct intercellular communications. Its expression is increased during lactation and reduced in obese mammary adipose tissue, which is resistant to lactation-induced remodeling. However, whether Cx43 is required for adipocyte remodeling and breastmilk production to support neonates' growth has not been established. In this study, we used doxycycline-inducible adipocyte-specific Cx43-deleted mice and demonstrated that adipocyte Cx43 played a vital role in determining the carbohydrate levels in breastmilk, which may subsequently affect neonates' growth.

The breast-feeding neonate depends on mother's milk for both macronutrients and micronutrients including minerals. The goals of the present study were to document the effects of genetic background in mice on milk concentrations of select minerals and to use genome-wide association study (GWAS) to identify quantitative trait loci (QTL) regulating milk mineral concentrations. Milk samples from lactating mice in each of 31 different inbred strains of the mouse diversity panel (MDP) were analyzed by inductively coupled plasma-optical emission spectroscopy to determine the concentrations of calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), phosphorus (P), sulfur (S), and zinc (Zn). GWAS identified a single pleiotropic milk mineral concentration QTL (Mmcq) on chromosome 3 for Ca, Mg, and P. For the remaining minerals, six QTL were detected for Fe, four for K, three for Zn, and one for S. Intersecting the Mmcq with published chromatin immunoprecipitation sequence data identified 15 out of 4633 high-linkage disequilibrium single-nucleotide polymorphisms that resided in signal transducer and activation of transcription 5 (STAT5) binding regions. A milk Fe-associated locus (Mmcq9) on chromosome 1 contained an SNP that localized to a STAT5 binding region and intersected with a HOMER motif predicted to bind the transcriptional regulator E74-Like ETS transcription factor 5. This locus also contained the genes for solute carrier family (Slc) members Slc9a2, Slc9a4, Slc39a10, and Slc40a1. Expression analysis of these transporters supports the conclusion that Slc9a2 and Slc40a1 within the mammary gland could mediate the effect of Mmcq9 on milk Fe concentration.

Breast cancer risk is intimately intertwined with exposure to estrogens. While more than 160 breast cancer risk loci have been identified in humans, genetic interactions with estrogen exposure remain to be established. Strains of rodents exhibit striking differences in their responses to endogenous ovarian estrogens (primarily 17β-estradiol). Similar genetic variation has been observed for synthetic estrogen agonists (ethinyl estradiol) and environmental chemicals that mimic the actions of estrogens (xenoestrogens). This review of literature highlights the extent of variation in responses to estrogens among strains of rodents and compiles the genetic loci underlying pathogenic effects of excessive estrogen signaling. Genetic linkage studies have identified a total of the 35 quantitative trait loci (QTL) affecting responses to 17β-estradiol or diethylstilbestrol in five different tissues. However, the QTL appear to act in a tissue-specific manner with 9 QTL affecting the incidence or latency of mammary tumors induced by 17β-estradiol or diethylstilbestrol. Mammary gland development during puberty is also exquisitely sensitive to the actions of endogenous estrogens. Analysis of mammary ductal growth and branching in 43 strains of inbred mice identified 20 QTL. Regions in the human genome orthologous to the mammary development QTL harbor loci associated with breast cancer risk or mammographic density. The data demonstrate extensive genetic variation in regulation of estrogen signaling in rodent mammary tissues that alters susceptibility to tumors. Genetic variants in these pathways may identify a subset of women who are especially sensitive to either endogenous estrogens or environmental xenoestrogens and render them at increased risk of breast cancer.

Mammalian glutaredoxin 3 (Grx3) has been shown to be important for regulating cellular redox homeostasis in the cell. Our previous studies indicate that Grx3 is significantly overexpressed in various human cancers including breast cancer and demonstrate that Grx3 controls cancer cell growth and invasion by regulating reactive oxygen species (ROS) and NF-κB signaling pathways. However, it remains to be determined whether Grx3 is required for normal mammary gland development and how it contributes to epithelial cell proliferation and differentiation in vivo. In the present study, we examined Grx3 expression in different cell types within the developing mouse mammary gland (MG) and found enhanced expression of Grx3 at pregnancy and lactation stages. To assess the physiological role of Grx3 in MG, we generated the mutant mice in which Grx3 was deleted specifically in mammary epithelial cells (MECs). Although the reduction of Grx3 expression had only minimal effects on mammary ductal development in virgin mice, it did reduce alveolar density during pregnancy and lactation. The impairment of lobuloalveolar development was associated with high levels of ROS accumulation and reduced expression of milk protein genes. In addition, proliferative gene expression was significantly suppressed with proliferation defects occurring in knockout MECs during alveolar development compared with wild-type controls. Therefore, our findings suggest that Grx3 is a key regulator of ROS in vivo and is involved in pregnancy-dependent mammary gland development and secretory activation through modulating cellular ROS.

Unlabelled: ᅟ: Starch requires six enzymes for digestion to free glucose: two amylases (salivary and pancreatic) and four mucosal maltase activities; sucrase-isomaltase and maltase-glucoamylase. All are deficient in suckling rodents., Objective: The objective of this study is to test (13)C-starch digestion before weaning by measuring enrichment of blood (13)C-glucose in maltase-glucoamylase-null and wild-type mice., Methods: Maltase-glucoamylase gene was ablated at the N-terminal. Dams were fed low (13)C-diet and litters kept on low (13)C-diet. Pups were weaned at 21 days. Digestion was tested at 13 and 25 days by intragastric feeding of amylase predigested (13)C-α-limit dextrins. Blood (13)C-glucose enrichment was measured by gas chromatography combustion isotope ratio mass spectrometry (GCRMS) using penta-acetate derivatives., Results: Four hours after feeding, blood (13)C-glucose was enriched by 26 × 10(3) in null and 18 × 10(3) in wild-type mice at 13 days and 0.3 × 10(3) and 0.2 × 103 at 25 days (vs. fasting p = 0.045 and p = 0.045). By jejunal enzyme assay, immunohistochemistry, or Western blots, there was no maltase activity or brush border staining with maltase-glucoamylase antibodies at 13 days, but these were fully developed in the wild-type mice by 25 days. In 13-day null mice, luminal contents were stained by maltase-glucoamylase antibodies. Lactating the mammary gland revealed maltase-glucoamylase antibody staining of alveolar cells. Reverse transcription/polymerase chain reaction (RT/PCR) of lactating glands revealed a secreted form of maltase-glucoamylase., Conclusions: (1) (13)C-α-limit dextrins were rapidly digested to (13)C-glucose in 13-day mice independent of maltase-glucoamylase genotype or mucosal maltase activity. (2) This experiment demonstrates that a soluble maltase activity is secreted in mouse mother's milk which enables suckling pup starch digestion well before brush border enzyme development. (3) This experiment with (13)C-α-limit dextrins needs to be repeated in human breast fed infants.

Milking frequency is known to affect milk production and lactation persistence in dairy cows. Despite this, the mechanisms underlying this effect are only partially understood. Previous work in dairy cows examining increases in milk yield due to increased milking frequency have identified changes in apoptosis and expression of genes regulating cytokine signaling. In addition, changes in mitochondrial biogenesis and function have been suggested to play a role during the lactation cycle in regulating milk production. The goal of this study was to test the hypothesis that, when maintained over an entire lactation, extreme differences in milking frequency would be reflected in differences in apoptosis, mammary mitochondrial number, and the mammary expression of genes known to inhibit cytokine signaling. Primiparous Holstein cows (n=6) were assigned to the study 40d before parturition after which 1 udder half was milked once daily (1×) and the other 4 times daily (4×) Mammary biopsies were collected at 15, 60, 120, and 230d of lactation. Average milk yield from the 4× side was 3 times higher than from the 1× side. Analysis of milk composition revealed that protein, lactose, and solids-not-fat percentages were lower in 1× than 4× udder halves. Mammary cell apoptosis was not affected by milking frequency. Mammary cell mitochondrial number, as estimated by succinate dehydrogenase staining, was higher in early lactation, decreasing as days in milk increased, and with increased milking frequency. Although mammary expression of α-lactalbumin (LALBA) and β-casein (CSN2) was significantly increased in 4× glands, the expression of suppressors of cytokine signaling were similar between 1×- and 4×-milked halves. These results support the conclusion that changes in milk production in response to extreme differences in milking frequency may be related to alterations in mitochondrial number and lactose synthesis, but not apoptosis., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Genetic background plays a dominant role in mammary gland development and breast cancer (BrCa). Despite this, the role of genetics is only partially understood. This study used strain-dependent variation in an inbred mouse mapping panel, to identify quantitative trait loci (QTL) underlying structural variation in mammary ductal development, and determined if these QTL correlated with genomic intervals conferring BrCa susceptibility in humans. For about half of the traits, developmental variation among the complete set of strains in this study was greater (P < 0.05) than that of previously studied strains, or strains in current common use for mammary gland biology. Correlations were also detected with previously reported variation in mammary tumor latency and metastasis. In-silico genome-wide association identified 20 mammary development QTL (Mdq). Of these, five were syntenic with previously reported human BrCa loci. The most significant (P = 1 × 10(-11)) association of the study was on MMU6 and contained the genes Plxna4, Plxna4os1, and Chchd3. On MMU5, a QTL was detected (P = 8 × 10(-7)) that was syntenic to a human BrCa locus on h12q24.5 containing the genes Tbx3 and Tbx5. Intersection of linked SNP (r(2) > 0.8) with genomic and epigenomic features, and intersection of candidate genes with gene expression and survival data from human BrCa highlighted several for further study. These results support the conclusion that mammary tumorigenesis and normal ductal development are influenced by common genetic factors and that further studies of genetically diverse mice can improve our understanding of BrCa in humans.

Significant variation exists for maternal nurturing ability in inbred mice. Although classical mapping approaches have identified quantitative trait loci (QTL) that may account for this variation, the underlying genes are unknown. In this study, lactation performance data among the mouse diversity panel were used to map genomic regions associated with this variation. Females from each of 32 inbred strains (n = 8-19 dams/strain) were studied during the first 8 days of lactation by allowing them to raise weight- and size-normalized cross-foster litters (10 pups/litter). Average daily weight gain (ADG) of litters served as the primary indicator of milk production. The number of pups successfully reared to 8 days (PNUM8) also served as a related indicator of maternal performance. Initial haplotype association analysis using a Bonferroni-corrected, genome-wide threshold revealed 10 and 15 associations encompassing 11 and 13 genes for ADG and PNUM8, respectively. The most significant of these associated haplotype blocks were found on MMU 8, 11, and 19 and contained the genes Nr3c2, Egfr, Sec61g, and Gnaq. Lastly, two haplotype blocks on MMU9 were detected in association with PNUM8. These overlapped with the previously described maternal performance QTL, Neogq1. These results suggest that the application of in silico QTL mapping is a useful tool in discovering the presence of novel candidate genes involved in determining lactation capacity in mice.

Lactose synthesis is believed to be rate limiting for milk production. However, understanding the molecular events controlling lactose synthesis in humans is still rudimentary. We have utilized our established model of the RNA isolated from breast milk fat globule from seven healthy, exclusively breastfeeding women from 6 h to 42 days following delivery to determine the temporal coordination of changes in gene expression in the carbohydrate metabolic processes emphasizing the lactose synthesis pathway in human mammary epithelial cell. We showed that milk lactose concentrations increased from 75 to 200 mM from 6 to 96 h. Milk progesterone concentrations fell by 65% at 24 h and were undetectable by day 3. Milk prolactin peaked at 36 h and then declined progressively afterward. In concordance with lactose synthesis, gene expression of galactose kinase 2, UDP-glucose pyrophosphorylase 2 (UGP2), and phosphoglucomutase 1 increased 18-, 10-, and threefold, respectively, between 6 and 72 h. Between 6 and 96 h, gene expression of UDP-galactose transporter 2 (SLC35A2) increased threefold, whereas glucose transporter 1 was unchanged. Gene expression of lactose synthase no. 3 increased 1.7-fold by 96 h, whereas α-lactalbumin did not change over the entire study duration. Gene expression of prolactin receptor (PRLR) and its downstream signal transducer and activator of transcription complex 5 (STAT5) were increased 10- and 2.5-fold, respectively, by 72 h. In summary, lactose synthesis paralleled the induction of gene expression of proteins involved in UDP-galactose synthesis and transport, suggesting that they are potentially rate limiting in lactose synthesis and thus milk production. Progesterone withdrawal may be the signal that triggers PRLR signaling via STAT5, which may in turn induce UGP2 and SLC35A2 expression.

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7-10 days.

The regulation of mitochondrial biogenesis and function in the lactating mammary cell is poorly understood. The goal of this study was to use proteomics to relate temporal changes in mammary cell mitochondrial function during lactation to changes in the proteins that make up this organelle. The hypothesis tested was that changes in mammary cell mitochondrial biogenesis and function during lactation would be accounted for by coordinated changes in the proteins of the electron transport chain and that some of these proteins might be linked by their expression patterns to PPARGC1α and AMP kinase. The mitochondrial proteome was studied along with markers of mitochondrial biogenesis and function in mammary tissue collected from mice over the course of a single prolonged lactation cycle. Mammary tissue concentrations of AMP and ADP were increased (P < 0.05) during early lactation and then declined with prolonged lactation. Similar changes were also observed for mitochondrial ATP synthesis activity, mitochondrial mass and DNA copy number. Analysis of the mammary cell mitochondrial proteome identified 244 unique proteins. Of these, only two proteins of the electron transport chain were found to increase during early lactation. In contrast, coordinated changes in numerous electron transport chain proteins were observed both during mid- and late lactation. There were six proteins that could be directly linked to PPARGC1α through network analysis. Abundance of PPARGC-1α and phosphorylation of AMP kinase was highest on day 2 postpartum. The results suggest that the increases in mammary mitochondria ATP synthesis activity during early lactation results from changes in only a limited number proteins. In addition, decreases in a handful of proteins linked to lipid oxidation could be temporally linked to decreases in PPARGC1α and phospho-AMP kinase suggesting potential roles for these proteins in coordinating mammary gland metabolism during early lactation.

Prematurity and overfeeding in infants are associated with insulin resistance in childhood and may increase the risk of adult disease. Total parenteral nutrition (TPN) is a major source of infant nutritional support and may influence neonatal metabolic function. Our aim was to test the hypothesis that TPN induces increased adiposity and insulin resistance compared with enteral nutrition (EN) in neonatal pigs. Neonatal pigs were either fed enteral formula orally or i.v. administered a TPN mixture for 17 d; macronutrient intake was similar in both groups. During the 17-d period, we measured body composition by dual-energy X-ray absorptiometry scanning; fasting i.v. glucose tolerance tests (IVGTT) and hyperinsulinemic-euglycemic clamps (CLAMP) were performed to quantify insulin resistance. On d 17, tissue was collected after 1-h, low-dose CLAMP for tissue insulin signaling assays. TPN pigs gained less lean and more body fat and developed hepatic steatosis compared with EN pigs. After 7 and 13 d, IVGTT showed evidence of insulin resistance in the TPN compared with the EN group. Fasting plasma glucose and insulin also were higher in TPN pigs. CLAMP showed that insulin sensitivity was markedly lower in TPN pigs than in EN pigs. TPN also reduced the abundance of the insulin receptor, insulin receptor substrate 1, and phosphatidylinositol 3 kinase in skeletal muscle and liver and the proliferation of total pancreatic cells and β-cells. Hepatic proinflammatory genes as well as c-Jun-N-terminal kinase 1 phosphorylation, plasma interleukin 6, and tumor necrosis factor-α were all higher in TPN pigs than in EN pigs. The results demonstrate that chronic TPN induces a hepatic inflammatory response that is associated with significant insulin resistance, hepatic steatosis, and fat deposition compared with EN in neonatal pigs. Further studies are warranted to establish the mechanism of TPN-induced insulin resistance and hepatic metabolic dysfunction and whether there are persistent metabolic consequences of this lifesaving form of infant nutritional support.

Previous work has indicated that respiratory activity of mitochondrial preparations prepared from lactating mammary tissue is often much lower than that of mitochondria isolated from other organs such as the liver. Initial studies in our own laboratory also found that mammary mitochondria prepared from lactating mice had much lower ATP synthesis activity than those isolated from liver tissue obtained from the same animals. In this paper, we describe an improved procedure for obtaining coupled mitochondria from the mammary tissue of lactating mice. Using a high-throughput assay for mitochondrial ATP synthesis, we demonstrated that mammary mitochondria, unlike liver mitochondria, are sensitive to the concentration of bovine serum albumin and to the choice of chelating agent used in the preparation and assay buffers. Mammary mitochondria prepared and assayed in buffers containing 1 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N' tetraacetic acid (EGTA) and 0.4% bovine serum albumin have a similar ATP synthesis activity as liver mitochondria. In addition, we show that the chelating agent EDTA ablates the ATP synthesis capacity of mammary mitochondria through a mechanism that does not involve the release of cytochrome c. We also demonstrate that these improved isolation and assay procedures are both scalable and applicable to bovine mammary tissue, and we describe optimal conditions for cryopreservation and recovery of functionally active mitochondria. This work will facilitate future studies aimed at determining the importance of mammary mitochondria to milk production.

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.

The molecular physiology underlying human milk production is largely unknown because of limitations in obtaining tissue samples. Determining gene expression in normal lactating women would be a potential step toward understanding why some women struggle with or fail at breastfeeding their infants. Recently, we demonstrated the utility of RNA obtained from breast milk fat globule (MFG) to detect mammary epithelial cell (MEC)-specific gene expression. We used MFG RNA to determine the gene expression profile of human MEC during lactation. Microarray studies were performed using Human Ref-8 BeadChip arrays (Illumina). MFG RNA was collected every 3 h for 24 h from five healthy, exclusively breastfeeding women. We determined that 14,070 transcripts were expressed and represented the MFG transcriptome. According to GeneSpring GX 9, 156 ontology terms were enriched (corrected P < 0.05), which include cellular (n = 3,379 genes) and metabolic (n = 2,656) processes as the most significantly enriched biological process terms. The top networks and pathways were associated primarily with cellular activities most likely involved with milk synthesis. Multiple sampling over 24 h enabled us to demonstrate core circadian clock gene expression and the periodicity of 1,029 genes (7%) enriched for molecular functions involved in cell development, growth, proliferation, and cell morphology. In addition, we found that the MFG transcriptome was comparable to the metabolic gene expression profile described for the lactating mouse mammary gland. This paper is the first to describe the MFG transcriptome in sequential human samples over a 24 h period, providing valuable insights into gene expression in the human MEC.

GH, prolactin (PRL), and IGF-I stimulate lactation-related metabolic processes in mammary epithelial cells. However, the ability of these factors to stimulate milk production in animals varies depending on species and experimental variables. Previous work in our laboratory demonstrated that transgenic overexpression of des(1-3)IGF-I within the mammary glands of lactating mouse dams increased lactation capacity during prolonged lactation. This work also suggested that some of the effects of the overexpressed IGF-I may have been mediated through elevated concentrations of IGF-I or PRL in the systemic circulation. In the present study, murine GH and PRL, and a human IGF-I analog, long-R3-IGF-I (LR3), were administered as s.c. injections to compare their ability to enhance milk production, and alter mammary gland signaling and gene expression. Lactation capacity, as measured by litter gain, was increased (P<0.05) by GH, but not by PRL. LR3 increased (P<0.05) mammary phospho-Akt and suppressors of cytokines signaling 3 (SOCS3) gene expression, and had a modest ability to increase (P<0.05) lactation capacity. GH both increased (P<0.05) mammary SOCS1 expression and decreased (P<0.05) mammary expression of tryptophan hydroxylase 1, the rate-limiting enzyme in the synthesis of serotonin and a potential feedback inhibitor of lactation. These results suggest that while both GH and IGF-I stimulate milk production in the lactating mouse, the effect of GH may be additionally mediated through IGF-I-independent effects associated with repression of mammary serotonin synthesis.

The insulin-like growth factor-1 (IGF-1) signaling axis is important for cell growth, differentiation and survival and increased serum IGF is a risk factor for prostate and other cancers. To study IGF-1 action on the prostate, we created transgenic (PB-Des) mice that specifically express human IGF-1(des) in prostate epithelial cells. This encodes a mature isoform of IGF-1 with decreased affinity for IGF binding proteins (IGFBP) due to a 3-amino acid deletion in the N terminus. Expression of IGF-1(des) was sufficient to cause hyperplastic lesions in all mice, however the well-differentiated lesions did not progress to adenocarcinoma within a year. Remarkably, crossing the PB-Des mice to an established model of prostate cancer delayed progression of organ-confined tumors and emergence of metastatic lesions in young mice. While dissemination of metastatic lesions was widespread in old bigenic mice we did not detect IGF-1(des) in poorly differentiated primary tumors or metastatic lesions. Expression of endogenous IGF-1 and levels of P-Akt and P-Erk were reduced independent of age. These data suggest that increased physiologic levels of IGF-1 facilitate the emergence of hyperplastic lesions while imposing a strong IGF-1-dependent differentiation block. Selection against IGF-1 action appears requisite for progression of localized disease and metastogenesis.

Little is known of the molecular regulation of human milk production because of limitations in obtaining mammary tissue from lactating women. Our objectives were to evaluate whether RNA isolated from breast milk fat globules (MFGs) could be an alternative to mammary biopsies and to determine whether intense breast pumping, which increases prolactin (PRL) secretion, will upregulate alpha-lactalbumin (alpha-LA, a major determinant of lactose synthesis) transcription. RNA was isolated from MFG and transcripts of interest were identified and quantitated by real-time RT-PCR using an external standard for normalization. In addition, we performed microarray studies to determine MFG RNA gene expression profile. Ten lactating women were studied using two protocols: protocol A with intense pumping from 0800 to 0814 h followed by short pumping and protocol B with intense pumping from 1200 to 1214 h preceded by short pumping. Plasma PRL and MFG alpha-LA mRNA expression were measured. During protocol A, plasma PRL (61+/-7-248+/-43 mug/l by 14 min) and alpha-LA (3.5+/-0.9 fold by 6 h; P<0.03) increased. During protocol B, PRL gradually increased over 4 h from 69+/-14 to 205+/-28 mug/l, and further to 329+/-23 mug/l by 12 min of intense pumping; alpha-LA mRNA expression did not increase significantly. We conclude that MFGs provide a unique source to study the in vivo regulation of gene expression in mammary epithelial cells. alpha-LA mRNA is abundant in the MFG and its expression may be regulated by hormonal and temporal factors.

P190-B RhoGAP (p190-B, also known as ARHGAP5) has been shown to play an essential role in invasion of the terminal end buds (TEBs) into the surrounding fat pad during mammary gland ductal morphogenesis. Here we report that embryos with a homozygous p190-B gene deletion exhibit major defects in embryonic mammary bud development. Overall, p190-B-deficient buds were smaller in size, contained fewer cells, and displayed characteristics of impaired mesenchymal proliferation and differentiation. Consistent with the reported effects of p190-B deletion on IGF-1R signaling, IGF-1R-deficient embryos also displayed a similar small mammary bud phenotype. However, unlike the p190-B-deficient embryos, the IGF-1R-deficient embryos exhibited decreased epithelial proliferation and did not display mesenchymal defects. Because both IGF and p190-B signaling affect IRS-1/2, we examined IRS-1/2 double knockout embryonic mammary buds. These embryos displayed major defects similar to the p190-B-deficient embryos including smaller bud size. Importantly, like the p190-B-deficient buds, proliferation of the IRS-1/2-deficient mesenchyme was impaired. These results indicate that IGF signaling through p190-B and IRS proteins is critical for mammary bud formation and ensuing epithelial-mesenchymal interactions necessary to sustain mammary bud morphogenesis.

Expression of insulin receptor substrates (IRS)-1 and -2 within the mammary gland was found to be high at mid-lactation and dramatically decreased with mammary involution. This observation supports the hypothesis that these proteins are induced in the mammary gland with lactogenesis and involved in normal milk synthesis. To test this hypothesis, lactation capacity, along with indices of mammary secretory cell glucose metabolism and cell signaling were compared in normal mice and mice carrying targeted mutations in either the Irs1 or Irs2 genes. Mammary IRS-1 and IRS-2 protein levels were increased within 1 day of parturition and reached maximal levels by 5 days post partum. Dams carrying germline mutations of Irs1 or Irs2 displayed reduced lactation capacity as assessed by weight gain of pup litters. The reduction was more dramatic in Irs1(-/-) versus Irs2(-/-) dams. Maternal body weight was also reduced in Irs1(-/-) dams as well as in Irs1(+/-) Irs2(+/-) dams. The loss of IRS-1 had little impact on mammary gland expression of milk protein mRNAs, glucose transport, or on the abundance and subcellular localization of hexokinases I and II. The loss of IRS-1 was associated with a compensatory increase in insulin-induced IRS-2 phosphorylation; however, the loss of IRS-1 did also cause a reduction in insulin-dependent mammary gland-specific activation of Akt phosphorylation. These results support the conclusion that IRS-1 is important for insulin-dependent activation of Akt signaling within the lactating mammary gland, but that loss of this protein has only modest impact on normal milk synthesis, since related signaling proteins such as IRS-2 may act in compensatory fashion.

Insulin receptor substrates (IRSs) are adaptor proteins that link signaling from upstream activators to multiple downstream effectors to modulate normal growth, metabolism, survival, and differentiation. Recent cell culture studies have shown that IRSs can interact with, and are functionally required for, the transforming ability of many oncogenes. Consistent with this, IRSs are elevated and hyperactive in many human tumors. IRSs respond to many extracellular signals that are critical for mammary gland development, and we have shown that IRSs disrupt normal mammary acini formation in vitro, and cause mammary tumorigenesis and metastasis in vivo. In this review we will discuss the role of IRSs in both transformation and cancer progression.

Insulin receptor substrates (IRSs) are signaling adaptors that play a major role in the metabolic and mitogenic actions of insulin and insulin-like growth factors. Reports have recently noted increased levels, or activity, of IRSs in many human cancers, and some have linked this to poor patient prognosis. We found that overexpressed IRS-1 was constitutively phosphorylated in vitro and in vivo and that transgenic mice overexpressing IRS-1 or IRS-2 in the mammary gland showed progressive mammary hyperplasia, tumorigenesis, and metastasis. Tumors showed extensive squamous differentiation, a phenotype commonly seen with activation of the canonical beta-catenin signaling pathway. Consistent with this, IRSs were found to bind beta-catenin in vitro and in vivo. IRS-induced tumorigenesis is unique, given that the IRSs are signaling adaptors with no intrinsic kinase activity, and this supports a growing literature indicating a role for IRSs in cancer. This study defines IRSs as oncogene proteins in vivo and provides new models to develop inhibitors against IRSs for anticancer therapy.

Mammary gland development is dependent upon the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis, this same axis has also been implicated in breast cancer progression. In this study we investigated the effect of a GH antagonist, pegvisomant (Somavert, Pfizer), on normal mammary gland development and breast cancer xenograft growth. Intraperitoneal administration of pegvisomant resulted in a 60% suppression of hepatic IGF-I mRNA levels and upto a 70-80% reduction of serum IGF-I levels. Pegvisomant administration to virgin female mice caused a significant delay of mammary ductal outgrowth that was associated with a decrease in the number of terminal end buds and reduced branching and complexity of the gland. This effect of pegvisomant was mediated by a complete inhibition of both GH and IGF-IR-mediated signaling within the gland. In breast cancer xenograft studies, pegvisomant caused shrinkage of MCF-7 xenografts, with an initial 30% reduction in tumor volume, which was associated with a 2-fold reduction in proliferation and a 2-fold induction of apoptosis. Long-term growth inhibition of MCF-7 xenografts was noted. In contrast, pegvisomant had no effect on MDA-231 or MDA-435 xenografts, consistent with primary growth of these xenografts being unresponsive to IGF-I both in vitro and in vivo. In MCF-7 xenografts that regressed, pegvisomant had only minor effects upon GHR and IGF-IR signaling. This data supports previous studies indicating a role for GH/IGF in mammary gland development, and suggests that pegvisomant maybe useful for the prevention and/or treatment of estrogen receptor positive breast cancer.

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.

Milk synthesis by the mammary gland declines during prolonged lactation despite the continued suckling stimulus and complete removal of mammary secretions. Although this process has been hypothesized to result from cellular aging there has been no reported analysis of aging markers in the lactating mammary gland. The goal of these studies was to relate lactation performance in the mouse during a single prolonged lactation cycle to changes in mammary development and mitochondrial oxidative damage. During an artificially prolonged lactation cycle, the capacity of the dams to support litter growth decreased over time. This decrease was associated with decreased mammary epithelial content. Cell proliferation, along with the percentage of mammary progenitor cells, was high during early lactation, but low during prolonged lactation. Apoptosis increased during prolonged lactation. Oxidative damage to mitochondrial DNA increased during the early postpartum period and remained elevated through the end of the cycle. In contrast oxidative damage to mitochondrial protein was high during early lactation and decreased through mid lactation to increase again with prolonged lactation. The results suggest that a single prolonged lactation cycle may replicate on an accelerated basis some of the changes that occur with a lifetime of aging in organs possessing more stable cell populations.

Background & Aims: Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive hormone that exerts diverse actions in the gastrointestinal tract, including enhancing epithelial cell survival and proliferation, mucosal blood flow, and nutrient uptake and suppressing gastric motility and secretion. These actions are mediated by the G-protein-coupled receptor, GLP-2R. Cellular localization of the GLP-2R and the nature of its signaling network in the gut, however, are poorly defined. Thus, our aim was to establish cellular localization of GLP-2R and functional connection to vascular action of GLP-2 in the gut., Methods: Intestinal cellular GLP-2R localization was determined with real-time, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of laser capture microdissected subtissue and fluorescence in situ hybridization and also with double and/or triple immunostaining of human and pig tissue using a validated GLP-2R polyclonal antibody. Superior mesenteric arterial blood flow and intestinal eNOS expression and phosphorylation were measured in TPN-fed pigs acutely (4 h) infused with GLP-2., Results: We show that the porcine GLP-2R mRNA was expressed in the villus epithelium and myenteric plexus. GLP-2R protein was co-localized by confocal immunohistochemistry with serotonin in enteroendocrine cells and also with endothelial nitric oxide synthase (eNOS)-expressing and vasoactive intestinal polypeptide-positive enteric neurons. In neonatal pigs, GLP-2 infusion dose-dependently stimulated intestinal blood flow and coordinately upregulated the expression of intestinal eNOS mRNA, protein, and phosphorylation (eNOS-Ser1117)., Conclusions: We conclude that the GLP-2-induced stimulation of blood flow is mediated by vasoactive neurotransmitters that are colocalized with GLP-2R in 2 functionally distinct cell types within the gastrointestinal tract.

During prolonged lactation, the mammary gland gradually loses the capacity to produce milk. In agricultural species, this decline can be slowed by administration of exogenous growth hormone (GH), which is believed to act through insulin-like growth factor 1 (IGF1). Our previous work demonstrated delayed natural mammary gland involution in des(1-3)IGF1-overexpressing transgenic mice (Tg[Wap-des{1-3}IGF1]8266 Jmr), hereafter referred to as WAP-DES mice. The present study tested the hypothesis that overexpressed des(1-3)IGF1 would delay the loss of milk production during prolonged lactation. Accordingly, we examined lactational performance in WAP-DES mice by artificially prolonging lactation with continual litter cross-fostering. Over time, lactational capacity and mammary development declined in both WAP-DES and control mice. However, the rate of decline was 40% slower in WAP-DES mice. Mammary cell apoptosis increased by 3-fold in both groups during prolonged lactation but was not different between genotypes. Plasma concentrations of murine IGF1 were decreased in WAP-DES mice, while those of the transgenic human IGF1 were elevated during prolonged lactation. Phosphorylation of the mammary IGF1 receptor was increased in the WAP-DES mice, but only during prolonged lactation. Plasma prolactin decreased with prolonged lactation in nontransgenic mice but remained high in WAP-DES mice. The WAP-DES mice maintained a higher body mass and a greater lean body mass during prolonged lactation. These data support the conclusion that overexpressed des(1-3)IGF1 enhanced milk synthesis and mammary development during prolonged lactation through localized and direct activation of the mammary gland IGF1 receptor and through systemic effects on prolactin secretion and possibly nutrient balance.

The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase that is essential to growth and development and also thought to provide a survival signal for the maintenance of the transformed phenotype. There has been increasing interest in further understanding the role of IGF-I signaling in cancer and in developing receptor antagonists for therapeutic application. We describe herein a novel animal model that involves transgenic expression of a fusion receptor that is constitutively activated by homodimerization. Transgenic mice that expressed the activated receptor showed aberrant development of the mammary glands and developed salivary and mammary adenocarcinomas as early as 8 weeks of age. Xenograft tumors and a cell line were derived from the transgenic animals and are sensitive to inhibition by a novel small-molecule inhibitor of the IGF-IR kinase. This new model should provide new opportunities for further understanding how aberrant IGF-IR signaling leads to tumorigenesis and for optimizing novel antagonists of the receptor kinase.

The mammalian genome is believed to contain some 30,000 to 40,000 different genes. Of these an estimated 42% have no known function. Genetically engineered mouse models (GEMM) have been a powerful tool available for determining gene function in vivo. In the mammary gland, a variety of genetic engineering approaches have been applied successfully to understanding the importance of specific gene products to mammary gland development and lactation. Our own laboratory has applied genetically engineered mice to facilitate understanding of the regulation of mammary gland development and lactation by insulin-like growth factors (IGF) and by the transcription factor, upstream stimulatory factor (USF-2). Our studies on transgenic mice that overexpress IGF-I have demonstrated the importance of IGF-dependent signaling pathways to maintenance of mammary epithelial cells during the declining phase of lactation. Our analysis of early developmental processes in mammary tissue from mice that carry a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential antiapoptotic effects. Lastly, our studies on mice that carry a targeted mutation of the Usf2 gene have demonstrated that this gene is necessary for normal lactation and have highlighted the importance of this gene to the maintenance of protein synthesis. These studies, as well as studies of others, have highlighted both the strengths and limitations inherent in the use of GEMM. Limitations serve as the driving force behind development of new experimental strategies and genetic engineering schemes that will allow for a full understanding of gene function within the mammary gland.

The detection of IGF-IR signaling in animal models has important implications for determining the role of this receptor in normal physiology and tumor growth. While many reports have correlated changes in plasma IGF-I levels in vivo with biological responses, few have shown that altered IGF-I levels can directly affect signaling within normal or tumor tissue. Here, we present new data that shows how the intravenous (IV) injection of IGF-I can be used to directly examine IGF signaling at the tissue level. Tail-vein IV injection of IGF-I into mice resulted in a rapid and dose-dependent activation of the IGF-I receptor and downstream phosphorylation of Akt and ERK1/2 in liver, kidney, and mammary gland. Similarly, IV IGF-I rapidly stimulated signaling in HT-29 colorectal and in MCF-7 breast cancer xenografts. This study shows how IV IGF injection can be used to examine the signaling mechanisms used by IGF-IR, in both normal mammary tissue and during tumor growth, and may provide a model for the characterization of IGF inhibitors.

Previous studies have suggested that upstream stimulatory factors (USFs) regulate genes involved with cell cycle progression. Because of the relationship of USFs to an important oncogene in breast cancer, c-myc, we chose to determine the importance of USF to normal mammary gland development in the mouse. Expression of USF in the mammary gland throughout development demonstrated only modest changes. Mutation of the Usf2 gene was associated with reduced fertility in females, but had no effect on prepartum mammary gland development. However, lactation performance in Usf2-/- females was only half of that observed in Usf2+/+ females, and both lactose and nitrogen were decreased in milk from Usf2-/- dams. This decrease was associated with diminished mammary tissue wet weight and luminal area by d 9 of lactation and with a decreased protein-DNA ratio. This decrease was associated with reduced abundance of the eukaryotic initiation factors eIF4E and eIF4G. Blood oxytocin concentrations on d 9 postpartum were also lower in Usf2-/- mice than Usf2+/+ mice. In contrast, the mutation had no effect on blood prolactin concentrations, mammary cell proliferation or apoptosis, mammary tissue oxytocin receptors, or milk protein gene expression. The mutation had only modest effects on maternal behavior. These data support the idea that USF is important to physiological processes necessary for the establishment and maintenance of normal lactation and suggest that USF-2 may impact lactation through both systemic and mammary cell-specific mechanisms.

E-cadherin is an important mediator of cell-cell interactions, and has been shown to play a crucial role in breast tumor suppression. Its inactivation occurs through instability at its chromosomal locus and mutations, but also through epigenetic mechanisms such as promoter hypermethylation and transcriptional silencing. We show here that the potent mitogen estrogen causes down-regulation of E-cadherin levels in both normal and tumorigenic breast epithelial cells, and that this down-regulation is reversed by antiestrogens. The reduction in E-cadherin levels is via a decrease in promoter activity and subsequent mRNA levels. Chromatin immunoprecipitation assays revealed that estrogen receptor and corepressors were bound to the E-cadherin promoter, and that overexpression of corepressors such as scaffold attachment factor B resulted in enhanced repression of E-cadherin. We propose that estrogen-mediated down-regulation of E-cadherin is a novel way of reducing E-cadherin levels in estrogen receptor-positive breast cancer.

Insulin receptor substrates (IRS) are central integrators of hormone, cytokine, and growth factor signaling. IRS proteins can be phosphorylated by a number of signaling pathways critical to normal mammary gland development. Studies in transgenic mice that overexpress IGF-I in the mammary gland suggested that IRS expression is important in the regulation of normal postlactational mammary involution. The goal of these studies was to examine IRS expression in the mouse mammary gland and determine the importance of IRS-1 to mammary development in the virgin mouse. IRS-1 and -2 show distinct patterns of protein expression in the virgin mouse mammary gland, and protein abundance is dramatically increased during pregnancy and lactation, but rapidly lost during involution. Consistent with hormone regulation, IRS-1 protein levels are reduced by ovariectomy, induced by combined treatment with estrogen and progesterone, and vary considerably throughout the estrous cycle. These changes occur without similar changes in mRNA levels, suggesting posttranscriptional control. Mammary glands from IRS-1 null mice have smaller fat pads than wild-type controls, but this reduction is proportional to the overall reduction in body size. Development of the mammary duct (terminal endbuds and branch points) is not altered by the loss of IRS-1, and pregnancy-induced proliferation is not changed. These data indicate that IRS undergo complex developmental and hormonal regulation in the mammary gland, and that IRS-1 is more likely to regulate mammary function in lactating mice than in virgin or pregnant mice.

Prolactin (PRL) is a major determinant of mammary epithelial cell proliferation during alveolar development in sexually mature and pregnant mice. To date, it has not been clear whether PRL effects these responses alone or by also invoking the action of autocrine/paracrine growth factors. In this study, we provide evidence that part of the effect of PRL on mammary gland growth is mediated by IGF-II. During sexual maturity and in early pregnancy, the level of IGF-II mRNA in the mammary gland was increased concurrent with increased PRL receptor expression. The level of IGF-II mRNA was reduced in mammary tissue from PRL receptor-/- mice during early pregnancy, and explants of mouse mammary gland and HC11 mammary epithelial cells both increased their expression of IGF-II after exposure to PRL in vitro. These findings coincided with the demonstration that IGF-II stimulated alveolar development in mammary glands in whole organ culture. PRL was most efficacious in stimulating IGF-II gene transcription from promoter 3 of the mouse IGF-II gene in vitro. Insight into the mechanism by which PRL induced IGF-II expression was provided by the fact that it was blocked by the Jak2 inhibitor AG490 and the MAPK inhibitor PD98059. Finally, induction of insulin receptor substrate (IRS)-1 in the mammary glands of PRL-treated mice and induction of IRS-1 and IRS-2 after treatment with PRL plus progesterone indicates that these molecules are induced by PRL as potential signaling intermediates downstream from IGF-I/insulin receptors. Together, these data demonstrate a role for IGF-II as a mediator of PRL action in the mouse mammary gland during ductal branching and alveolar development.

The explosion of signal transduction research over the last 10 years has provided a unique insight into the complexity of these intricate pathways. Whereas intermediates of multiple signaling pathways have been identified, understanding their function and, in particular, the interactions between them has become a daunting task. The increasing evidence that many of these pathways can cross-talk with each other via signal transactivation inevitably raises the question of how cells determine specificity in signaling. Despite the mind-numbing complexity of these pathways, progress has been made in developing highly specific and potent signal transduction inhibitors (STIs). STIs show promise in the treatment of cancer in preclinical studies and are currently in a number of clinical trials. Whereas many of these agents were "rationally designed," we barely understand their mechanisms of action. This review will highlight how recent studies using these STIs have elucidated novel mechanisms of STI action that may be used in the development of new therapeutic strategies for the treatment of cancer.

Insulin-like growth factors (IGF) are now known to play an important role in normal mammary gland development and have been implicated as risk factors in the etiology of breast cancer. Studies in genetically engineered mouse models have demonstrated that the IGF system acts within the mammary epithelium at key developmental stages to either stimulate cell cycle progression or inhibit cell death. The importance of these two actions to mammary gland development varies with the different developmental stage. Cell cycle effects appear to predominate during virgin ductal development and early pregnancy while cell survival effects are important for initiation and maintenance of lactation. This variation with stage of development most likely reflects changes in specific populations of epithelial cells within the gland. Relatively little information exists however on the actions of the IGF system during fetal or very early postnatal development. In addition much remains to be learned about the intracellular signaling mechanism that mediate the effects of IGFs on the different populations of epithelial cells present within the gland. This review summarizes the current state of knowledge concerning IGF actions, and mechanisms of action, within the normal mammary gland.

Insulin-like growth factor I (IGF-I) is known to regulate mammary gland development. This regulation occurs through effects on both cell cycle progression and apoptosis. Our laboratory has studied the IGF-I-dependent regulation of these processes by using transgenic and knockout mouse models that exhibit alterations in the IGF-I axis. Our studies of transgenic mice that overexpress IGF-I during pregnancy and lactation have demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation but has minimal effects during early lactation on milk composition or lactational capacity. In contrast, our analysis of early developmental processes in mammary tissue from mice carrying a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential anti-apoptotic effects. With both models, the effects of perturbing the IGF-I axis are dependent on the physiological state of the animal. The diminished ductal development that occurs in response to loss of the IGF-I receptor is dramatically restored during pregnancy, whereas the ability of overexpressed IGF-I to protect mammary cells from apoptosis does not occur if the mammary gland is induced to undergo forced involution. Data from our laboratory on the expression of IGF-signaling molecules in the mammary gland suggest that this effect of physiological context may be related to the expression of members of the insulin receptor substrate family.

IGF-I mediates mammary ductal development through stimulation of terminal end bud (TEB) development; however, no published data exist on the mechanism through which this occurs. The mechanism of IGF-I action on the TEB was studied by determining the requirement for the IGF-I receptor (IGF-IR) in IGF-I-dependent ductal development. We hypothesized that loss of the IGF-IR would disrupt mammary ductal development through a combination of decreased proliferation or increased apoptosis. Because IGF-IR null mice die at birth, embryonic mammary gland transplantation was used to study the effects of a disrupted IGF-IR gene. Analyses of grafts after 4 or 8 wk of development demonstrated a limited growth potential of the null mammary epithelium in virgin hosts. Bromodeoxyuridine labeling and terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling showed that cell proliferation was significantly decreased in null TEBs, but apoptosis was not. In addition, both the size and number of TEBs were reduced in null outgrowths. In pregnant hosts, null ductal growth was stimulated beyond the level seen in virgin hosts. These findings directly establish a proliferation-dependent role for the IGF-IR in the cells of the TEB. Additionally, this study indicates that pregnancy-dependent compensatory mechanisms can stimulate mammary development in the absence of an IGF-IR.

Overexpression of des(1-3) human insulin-like growth factor I (IGF-I) in the mammary glands of transgenic mice (WAP-DES) inhibits apoptosis during natural, but not forced, mammary involution. We hypothesized that this differential response would correlate with the expression of IGF signal transducers. Forced and natural involution were analyzed in nontransgenic and WAP-DES mice beginning on day 16 postpartum. During natural involution, mammary gland wet weight was higher and apoptosis was lower in WAP-DES than in nontransgenic mice. The WAP-DES transgene had no effect on these parameters during forced involution. Mammary tissue concentrations of the transgene protein were 2- to 10-fold higher than those of endogenous IGF-I. Western blot analysis of pooled mammary tissue extracts demonstrated only slightly higher phosphorylation of the IGF signal transducers insulin receptor substrate-1 (IRS-1) and Akt in the WAP-DES than in nontransgenic mice. Dramatic early reductions in phospho-IRS-1, phospho-Akt, IRS-1, IRS-2, and Akt proteins occurred during forced, but not natural, involution. The abundance of the IGF-I receptor and the messenger RNAs for the IGF-I receptors, IRS-1 and -2, were not affected by either genotype or involution. These findings support the conclusions that mammary cells lose their responsiveness to insulin-like signals during forced involution, and that posttranscriptional or posttranslational regulation of IRS-1 and IRS-2 may play a role in this loss.

Correct temporal and spatial regulation of apoptosis is critical for normal mammary gland development and lactation. Previous work with a strain of transgenic mice that overexpress des(1-3)hIGF-I during pregnancy and lactation suggested that this growth factor inhibits apoptosis. The hypothesis tested within these studies is that overexpression of des(1-3)hIGF-I within the mammary gland inhibits apoptosis and the expression of apoptosis-associated genes that are known to be activated by the transcription factor AP-1. This inhibition of apoptosis was further posited to predispose the tissue to carcinogenesis. TUNEL analysis of mammary tissue from transgenic mice that overexpress des(1-3)hIGF-I under control of the rat whey acidic protein promoter showed only 25% (P < 0.05) of the number of apoptotic cells found in nontransgenic mice at the same stage of lactation. Northern analysis of RNA from these animals showed a 75% (P = 0.08) reduction in c-Jun mRNA abundance. Histological analysis of mammary tissue from nonlactating multiparous WAP-DES mice ranging in age from 13 to 25 months showed a variety of hyperplastic lesions. These lesions aberrantly expressed the transgene. At 23 months of age 50% of the transgenic mice within this study developed adenocarcinomas. These results support the conclusion that inhibition of apoptosis within the mammary gland by IGF-I involves decreased activity of AP-1 and predisposes the tissue to tumors.

Mammary tumorigenesis was analysed in transgenic mice which overexpress des(1-3)hIGF-I (WAP-DES) and/or a mutant form of p53 (p53172R-H). Nonlactating, multiparous WAP-DES mice exhibited hyperplastic lesions termed mammary interepithelial neoplasia (MIN) which constitutively expressed WAP-DES. By 23 months of age, 53% of the WAP-DES mice developed mammary adenocarcinomas. A 75% reduction in both apoptosis and proliferation was observed in the normal mammary glands of WAP-DES mice. Mammary tumor incidence in WAP-DES/p53 bitransgenic mice was similar to that of WAP-DES and 2 - 3-fold greater than that of nontransgenic and p53172R-H females. Tumor latency, however, was reduced by 8 months in bitransgenic mice as compared to mice of the other three genotypes. Aneuploidy was frequently observed in tumors from bitransgenic and p53172R-H mice, but not from mice expressing only the WAP-DES transgene. Expression of IGFBP3 was elevated in tumors from WAP-DES, but not bitransgenic mice, indicating an alteration in the p53/IGF-I axis. These studies indicate that overexpression of des(1-3)hIGF-I increases the frequency of MIN and stochastic mammary tumors and that the appearance of tumors displaying genomic instability is accelerated by mutant p53172R-H. Oncogene (2000) 19, 889 - 898.

Transgenic and knockout mice have become valuable experimental systems with which to study specific molecular events within the mammary gland of an intact animal. These models have provided a wealth of information about the effects of a number of oncogenes and growth factors. This review focuses on results obtained from the application of transgenic and knockout models to determine the roles of insulin and insulin-like growth factors (IGF) in the regulation of mammary gland development, lactation and tumorigenesis. Transgenic models which overexpress IGF-I or -II display specific alterations in mammary gland development and an increased incidence of mammary tumors. Analysis of mammary gland development in knockout mice which are deficient in IGF-I or the IGF-I receptor supports the conclusion that the IGF system is important for normal mammary gland development. This review discusses these observations in detail and attempts to fit them into a larger picture of IGF and insulin action in the mammary gland.

We tested the hypothesis that chronic ingestion of increased concentrations of milk-borne des(1-3) human insulin-like growth factor-I (hIGF-I) stimulates gastrointestinal growth and development in suckling mice. We used a transgenic mouse with targeted, lactation-dependent, overexpression of des(1-3) hIGF-I in the mammary gland (IGF). Pups were suckled (7 pups per litter) from birth by either IGF (n = 3-6 litters) or control (n = 3-5 litters) dams. In IGF and control pups, we measured the growth (protein and DNA content) and protein synthesis rate (3H-phenylalanine incorporation) of gastrointestinal and visceral organs in 4-, 8-, 12-, 16- and 29-d-old pups. Des(1-3) hIGF-I in milk from IGF dams was 40-200-fold higher than mouse IGF in either IGF or control dams, but was not detected in the plasma of pups suckling IGF dams. Small intestinal weight, protein and DNA content at 8 and 16 d were greater in pups suckling IGF dams than control dams; protein synthesis was also greater in IGF pups at 8 d. Total intestinal lactase activity at 8 and 12 d of age tended to be higher (P < 0.10) in IGF than in control pups. Hypersecretion of des(1-3) hIGF-I in milk ingested by suckling mice pups had limited effects on the growth and maturation of the gastrointestinal tract. Moreover, there was little evidence that milk-borne IGF-I is absorbed into the circulation and stimulates visceral organ growth. This study also demonstrates the feasibility of using mammary-specific transgenes to increase the concentration of milk-borne growth factors to examine whether they affect the growth and development of the suckling neonate.

To test the hypothesis that insulin-like growth factor I (IGF-I) regulates mammary gland development and lactation, the expression of both human (h) IGF-I and des(1-3)hIGF-I was targeted to the mammary gland in transgenic mice using a novel exon replacement strategy and the rat whey acidic protein (rWAP) gene regulatory sequences. Both transgenes expressed a 0.7-kilobase messenger RNA (mRNA). The abundance of WAP-IGE-I and WAP-DES mRNA on day 10 of lactation ranged from 0.2-1.0% and 0.2-13% of the endogenous mouse WAP mRNA, respectively. For WAP-DES mice, transgene expression was greatest from midpregnancy throughout lactation. Western blot analysis showed the presence of correctly processed hIGF-I in milk from these transgenic mice. This hIGF-I was capable of stimulating protein synthesis in cultured rat L6 myoblasts. Ligand blotting indicated changes in mammary gland secretion of IGFBP in response to WAP-DES expression. Histological analysis of mammary tissue from mice overexpressing des(1-3)hIGF-I showed incomplete mammary involution, ductile hypertrophy, and loss of secretory lobules associated with increased deposition of collagen. These changes are believed to occur through autocrine and paracrine effects of des(1-3)-hIGF-I on both epithelial and stromal cells.

The goal of this study was to relate insulin-like growth factor (IGF) binding with IGF-stimulated growth in a murine mammary epithelial (COMMA-D/MME) cell line. Affinity crosslinking with [125I]IGF-I showed major bands at 224,000 and 148,000 Mr that were ablated by the inclusion of IGF-I or -II at 130 nM. Scatchard-transformed [125I]IGF-I binding data was best fit by a two-site model, with 24,000 sites possessing a Kd of 0.33 nM and 1,000 sites possessing a Kd of 8.09 nM per cell. Competition analysis showed ED50 values for IGF-I, -II, and insulin to be 0.90 +/- 0.03, 7.15 +/- 4.27, and 335 +/- 104 nM, respectively. Affinity crosslinking of [125I]IGF-II showed three major bands of 230,000, 148,000, and 61,000 to 58,000 Mr. Unlabeled IGF-II ablated all bands, while IGF-I and insulin ablated only the 148,000 Mr band. Competition analysis showed ED50 values for unlabeled IGF-I and -II to be 0.10 +/- 0.01 and 5.31 +/- 2.04 nM, respectively. In spite of the receptor affinity differences, no significant difference was noted in IGF-I and -II in capacity to stimulate cell growth. These data indicate that COMMA-D/MME cells express IGF receptors and that both IGFs are mitogenic.

Colostrum is rich in IGF-I and IGF-II, and the dietary effects of recombinant human (rh)IGF-I on the newborn are of interest. The objective of this study was to examine the effects of dietary rhIGF-I on intestinal tissue growth and populations of IGF receptors. Twenty-three male diary calves were fed one of three experimental diets: 1) milk replacer plus isolated colostrum-derived globulins (MR-), 2) same as 1 plus 750 ng of rhIGF-I/mL (MR+), or 3) pooled cow colostrum (COL). After the first four feedings, all calves received milk replacer without additional globulins; calves fed the MR+ diet continued to receive the addition of 750 ng of rhIGF-I/mL until the experiment ended at 7 d after birth. Calves were killed and intestinal tissue was collected for in vitro [3H]thymidine incorporation studies. Incorporation differed among intestinal regions (duodenum, jejunum, and ileum). The MR+ calves had greater (P < .01) [3H]thymidine incorporation per unit of DNA than either the COL or MR- calves (31.8 vs 18.6 and 11.5 x 10(3) dpm/microgram of DNA, respectively). Competitive binding analysis indicated the presence of specific type 1 and type 2 intestinal IGF receptors. The IGF-I was more potent than IGF-II and insulin at inhibiting [125I]rhIGF-I binding (ED50 was 1.84, 9.17, and 1.91 ng/mL, respectively). The IGF-II was the only ligand capable of inhibiting [125I]rhIGF-II binding (ED50 was .30 nmol/mL).(ABSTRACT TRUNCATED AT 250 WORDS)

The objectives of these studies were to determine if the concentration of insulin-like growth factor-I (IGF-I) in mammary colostrum secretions could be altered through manipulation of IGF-I concentrations in blood and to compare the temporal changes of IGF-I in mammary secretions to those occurring for IgG1. Milking of 15 pregnant Holstein cows was stopped at 8 weeks prepartum and they were randomly assigned to one of three treatments. A control (C) treatment consisted of feeding the animals 100% of NRC requirements for protein and energy. A second group of cows was fed as the control group and injected with 1.8 mumol bovine GH/day. The third group was fed at 70% of NRC requirements for protein and energy to cause a moderate nutrient restriction (NR). Body weight was measured weekly. Blood was collected by tail venepuncture at 4 h intervals for 24 h. Mammary secretions were collected and pooled among contralateral front and rear quarters (diagonal) for measurement of volume, IGF-I and IgG1 concentrations. Samples were collected at -7, -5, -2, 0 and 1 week postpartum. Cows on the NR treatment failed to gain weight during the dry period compared with C cows (P < 0.05). Blood GH and IGF-I concentrations (P > 0.1) were unaffected by NR treatment. Cows treated with GH had higher (P < 0.01) serum GH and IGF-I levels throughout the entire treatment period, and higher serum IgG1 at 5 and 2 weeks prepartum (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of IGF-I and IGF-II, and IGF binding proteins (IGFBP) in serum and mammary gland secretions were surveyed during the dry period and early lactation of 30 Holstein cows. Although there was a threefold drop in the concentration of IGF-I in serum from the last week of the dry period to parturition (81 +/- 7 to 24 +/- 3 ng/ml, P less than .01), there was no significant change in serum IGF-II concentration during this period (150 +/- 17 vs 173 +/- 13 ng/ml, P greater than .05). Furthermore, a 57% increase in serum IGF-I was observed from the last week of lactation to the second week of drying off (100 +/- 5 to 157 +/- 8 ng/ml, P less than .05). Changes in serum IGF-II were not observed (126 +/- 11 vs 150 +/- 10 ng/ml, respectively; P greater than .05). Although IGF-I, IGF-II, and IGFBP concentrations in mammary secretions peaked 2 wk before parturition (2.95 +/- 1.1, 1.83 +/- .6, and 7.27 +/- .76 micrograms/ml, respectively), total output/quarter was highest in colostrum (394 +/- 119, 295 +/- 132, and 2,680 +/- 1,967 micrograms/quarter, respectively). Weekly milking of two individual quarters during the dry period did not affect (P greater than .05) IGF-I or IGF-II concentration (ng/ml) or total output (microgram/quarter) and milk yield in colostrum and milk (2 wk and 7 wk) compared with the ipsilateral quarter. The data support the hypothesis that IGF-I may be transported by the mammary gland epithelium. Furthermore, the secretion mechanisms of IGF-I, IGF-II, and IGFBP by the gland may be related to each other.