Journal of Dairy Science
Volume 91, Issue 11 , Pages 4183-4189, November 2008

Leptin Up-Regulates the Lactogenic Effect of Prolactin in the Bovine Mammary Gland In Vitro

  • Y. Feuermann

      Affiliations

    • Institute of Animal Science, Agricultural Research Organization, the Volcani Center, PO Box 6, Bet Dagan 50250, Israel
    • ,
  • A. Shamay

      Affiliations

    • Institute of Animal Science, Agricultural Research Organization, the Volcani Center, PO Box 6, Bet Dagan 50250, Israel
    • ,
  • S.J. Mabjeesh

      Affiliations

    • Department of Animal Science, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel
    • Corresponding Author InformationCorresponding author.

Received 2 January 2008; accepted 13 June 2008.

Article Outline

Abstract 

The ability of leptin to up-regulate prolactin action in the mammary gland is well established. We examined the effect of leptin and prolactin on traits associated with lactation. Leptin and prolactin enhanced proliferation (thymidine incorporation) of the mammary gland cells, elevated the cells’ proliferation in a dose-responsive manner, and synergized to elevate the expression of amino acid metabolism via a 90% increase in aminopeptidase N expression. Leptin and prolactin decreased apoptosis (decreased caspase-3 expression by 60%) in the same manner. Leptin enhanced the effect of prolactin on all of these processes in bovine mammary explants. Leptin and prolactin regulated mTOR (mammalian target of rapamycin) by increasing expression by 66%, which is one of the signal-transduction junctions involved in the regulation of proliferation, apoptosis, and protein synthesis. These findings support the hypothesis that leptin up-regulates prolactin action in the bovine mammary gland.

Key words: leptin, prolactin, apoptosis, aminopeptidase N/CD13

 

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Introduction 

The initiation of lactation in dairy cows requires a complex interaction of hypophyseal, adrenal, ovarian, and placental hormones (Akers, 1985). In recent years, the mechanisms governing hormonal control have begun to be unraveled. Leptin up-regulated the lactogenic effect of prolactin in the bovine mammary gland (Feuermann et al., 2004). The importance of prolactin to bovine lactation is not clear. It was shown that CB154, a synthetic ergot alkaloid that inhibits prolactin secretion, lowered prolactin concentration by 80%, prevented the normal periparturient increase in serum prolactin, and reduced milk production, but did not abolish lactation in treated cows (Akers et al., 1981). Moreover, prolactin induced maximal synthesis of casein in explants prepared from lactating bovine mammary gland (Gertler et al., 1982). The initiation phase of lactation is characterized by upregulation of milk production and cell proliferation and a decrease in the apoptotic process in bovine mammary gland (Stefanon et al., 2002). One of the factors affecting these processes is prolactin. Prolactin binds to its receptor in the mammary gland and activates several pathways, including the Janus kinase/signal transducers and activators of transcription pathway. These genes are associated with proliferation, differentiation, and lactogenesis (Hennighausen et al., 1997).

Leptin expression was found in mammary adipocytes of sheep during the early phase of pregnancy and in mammary epithelial cells during lactation (Bonnet et al., 2002). Leptin receptors are present in 6 differently spliced isoforms named Ob-Ra to Ob-Rf (Sweeney, 2002). The tissue distribution of the leptin receptor isoforms is not well established, and the expression of Ob-Ra has only been reported in the adrenal gland (Yanagihara et al., 2000), pituitary gland, liver, and spleen of cattle (Chelikani et al., 2003), and in lactating ovine mammary glands (Laud et al., 1999). Expression of Ob-Ra was recently found in adipose tissue of Holstein dairy cattle (Bernabucci et al., 2006). Thorn et al. (2006) demonstrated that leptin does not have a direct effect on mammary epithelial cells from prepubertal dairy heifers. Indeed, we demonstrated that leptin alone had no effect on bovine lactating mammary gland, whereas in the presence of prolactin, leptin amplified prolactin's action on mature lactating bovine mammary gland (Feuermann et al., 2004). In this study, we examined other aspects of the interaction between leptin and prolactin. The objective focused on the effect of the interaction between leptin and prolactin on the processes related to the initiation of lactation.

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Materials and Methods 

Animals 

Mammary tissue was obtained from Holstein cows (2nd and 3rd parity) in the slaughterhouse, 2 to 5 mo after the onset of lactation, under veterinarian supervision. All procedures were approved by the Israeli Ministry of Agriculture in accordance with Israeli regulations for animal experimentation.

Materials 

Culture medium M-199, penicillin, streptomycin, and Fungizone (amphotericin B) were obtained from Bet HaEmek, Israel (Biological Industries, Bet HaEmek, Israel). Bovine insulin, cortisol, and ovine prolactin were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Mammary Explant Culture 

Mammary tissue and mammary fat tissue obtained from cows in the slaughterhouse were transferred to the laboratory in M-199 medium containing 100 U of penicillin, 100μg of streptomycin, 0.25μg of Fungizone, and 1μg/mL insulin. Explants were prepared as described previously (Shamay et al., 1987). Twenty explants (50 to 80mg total weight) were placed on an impregnated lens paper floating in 5mL of M-199 medium. For the first 3 d, the mammary explants were incubated in medium supplemented with insulin and cortisol, and then prolactin and leptin were added according to the experimental protocol as follows. Explants from lactating bovine mammary gland were incubated with 1μg/mL insulin and 0.5μg/mL cortisol for 3 d; after 3 d the medium was supplemented with leptin, prolactin, or both, for another 2 d. Prolactin was included at 0, 0.1, and 1μg/mL, whereas leptin was included at 0, 1, 10, 50, and 100ng/mL. The medium was changed every 24h for 5 d. Each treatment within experiment was performed in 3 replicates.

Western Blot Analysis 

Samples (40μg of protein in 20μL of buffer) were boiled in 3× loading buffer (10mM Tris-HCl, pH 6.8, including 3% wt/vol SDS, 5% wt/vol β-mercaptoethanol, 20% wt/vol glycerol, and 0.6% wt/vol bromophenol blue) for 3min, separated in a 12% Bis-Tris gel under reducing conditions (1h at 190V), and transferred to nitrocellulose membranes (Millipore PVDF, 0.45μm, Millipore, Bedford, MA). For blocking, membranes were incubated in PBS with 0.05% (wt/vol) Tween-20 (PBS-T) and 1% (wt/vol) nonfat dry milk overnight. The membranes were washed in PBS-T and incubated for 75min with the specified primary antibody. After washing again in PBS-T, the membranes were incubated with horseradish peroxidase-linked secondary antibody for 45min. They were then washed again in PBS-T and incubated in enhanced chemiluminescence reagent (ECL Western Blotting Analysis System, Amersham-Pharmacia, Buckinghamshire, UK) for 30s. Primary and secondary antibodies are given in Table 1.

Table 1. Primary and secondary antibodies used for the Western blot analyses1
Primary antibodyDilutionSecondary antibodyDilution
Anti-CD-13 (c-17) goat polyclonal1:500HRP-anti-goat anti-mouse IgG conjugated1:20,000
IgG (cat no. sc-6995)
Anti-caspase 3/apopain rabbit polyclonal IgG (cat no. 06-529)1:500Donkey anti-rabbit IgG (H&L), HRP1:10,000
Anti-cleaved caspase-3 (Asp175) (cat no. 9661s)1:1,000Donkey anti rabbit IgG (H&L), HRP1:10,000
Rabbit anti-mTOR (cat no. 38-9500)1:50Donkey anti-rabbit IgG (H&L), HRP1:10,000
Monoclonal anti-BSA clones BSA-33 (cat no. B-2901)1:250Anti-mouse IgG (whole molecule) peroxidase-conjugated (cat no. A-4416)1:2,500

1Source: Anti-CD-13 was from Santa Cruz Biotechnology (San Francisco, CA); HRP-anti-goat anti-mouse IgG and rabbit anti-mTOR were from Zymed (San Francisco, CA); anti-caspase 3/apopain rabbit polyclonal IgG was from Upstate (Lake Placid, NY); donkey anti-rabbit IgG and horseradish peroxidase (HRP) were from Promega (Madison, WI); anti-cleaved caspase-3 was from New England Biolabs (Ipswich, MA); monoclonal anti-BSA clones BSA-33 and anti-mouse IgG were from Sigma-Aldrich Corp. (St. Louis, MO).

In Vitro Incorporation of [3H]Thymidine 

Explants were prepared from bovine mammary glands and labeled in vitro with [3H]thymidine for 90min as described previously (Iavnilovitch et al., 2002).

Statistical Analysis 

Statistical analyses of all data and [3H]thymidine incorporation were carried out by one-way ANOVA using the JMP 5.0.1 statistical package from SAS (SAS Institute, 2001). Results were considered different at P<0.05. Results are presented as means±SE.

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Results 

Effect of Leptin and Prolactin on Albumin Synthesis in Bovine Mammary Gland Explants 

Leptin alone in the culture medium did not affect albumin expression in the explants (Figure 1). Prolactin alone did not have a dramatic effect on albumin expression; however, 1μg/mL prolactin together with 100ng/mL leptin elevated (P<0.01) albumin accumulation to its greatest level (Figure 1).

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  • Figure 1. 

    Effect of leptin (L; 0, 1, 10, and 100ng/mL) and prolactin (P; 0 or 1μg/mL) in the presence of insulin (I; 1μg/mL) on albumin synthesis in bovine mammary gland explants. Western blot (WB) analysis of albumin from explants of lactating bovine mammary glands shows that leptin alone increased albumin accumulation in the explants. The greatest accumulation of albumin was observed when prolactin at 1μg/mL and leptin at 100ng/mL were introduced together into the medium. Results are least squares means of the scanning results ± SE (n=3, P<0.01, SEM=0.12). A,BBars with different letters differ (P<0.05).

Effect of Leptin and Prolactin on Proliferation in Bovine Mammary Gland Explants 

Cell proliferation was evaluated by [3H]thymidine incorporation. Prolactin at 0.1μg/mL in the medium did not affect proliferation in the mammary explants, whereas at 1μg/mL, it up-regulated proliferation (Figure 2). Leptin at 50ng/mL did not affect cell proliferation in the explants. When combined with prolactin, leptin elevated cell proliferation in a dose-responsive manner, with the greatest rate of proliferation observed when 100ng/mL leptin was added to medium containing 1μg/mL prolactin (Figure 2).

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  • Figure 2. 

    Effect of leptin (0, 10, 50, and 100ng/mL) and prolactin (0, 0.1, and 1μg/mL) on DNA synthesis in bovine mammary gland explants. Cell DNA synthesis was evaluated by [3H]thymidine incorporation. Explants from lactating bovine mammary gland were incubated with leptin, prolactin, or both. Prolactin at 1μg/mL up-regulated DNA synthesis. Leptin at 50ng/mL did not affect DNA synthesis in the explants, but leptin in the presence of prolactin elevated the DNA synthesis in a dose-responsive manner. The greatest rate of DNA synthesis was observed with 1 mg/mL prolactin combined with 100ng/mL leptin. Results are least squares means ± SE (n=3). A-DBars with different letters differ (P<0.05).

Effect of Leptin and Prolactin on Apoptosis in Bovine Mammary Gland Explants 

Caspase-3 cleaved and uncleaved protein expression was analyzed by Western blot (Figure 3). Caspase-3 expression was down-regulated with addition of 1μg/mL prolactin to medium containing 100ng/mL leptin compared with treatments with leptin alone.

  • View full-size image.
  • Figure 3. 

    Effect of leptin (0, 10, and 100ng/mL) and prolactin (0 and 1μg/mL) on apoptosis (caspase-3) in bovine mammary gland explants. Western blot (WB) analysis of caspase-3 protein expression was down-regulated by 60% with addition of 1μg/mL prolactin to a culture containing 100ng/mL leptin in the medium. Results are least squares means of the scanning results ± SE (n=3). A,BBars with different letters differ (P<0.05).

Leptin and Prolactin Regulate Aminopeptidase N/CD13 Expression in Bovine Mammary Gland Explants 

Aminopeptidase N/CD13 (APN) protein expression, as determined by Western blot analysis, after 24h of incubation with the different treatments is shown in Figure 4. Addition of leptin to medium containing prolactin up-regulated (P<0.01) APN expression to its greatest level.

  • View full-size image.
  • Figure 4. 

    Leptin (L; 0 and 100ng/mL) and prolactin (P; 0 and 1μg/mL) regulation of aminopeptidase N/CD13 (APN) expression in bovine mammary gland explants in the presence of insulin (I; 1μg/mL). Western blot (WB) analysis showed that leptin combined with prolactin upregulated the expression of APN by approximately 90%. Results are least squares means of the scanning results ± SE (n=3; P<0.001, SEM = 0.11). A,BBars with different letters differ (P<0.05).

Leptin and Prolactin Regulate Mammalian Target of Rapamycin Protein Level in Bovine Mammary Gland Explants 

Western blot analysis of mammalian target of rapamycin (mTOR) protein level is shown in Figure 5. Leptin alone had no effect on mTOR protein level in the explants. Medium containing 1μg/mL prolactin did not elevate mTOR protein above that induced by leptin alone. The greatest expression (∼66%; P<0.05) of mTOR was achieved when leptin and prolactin were introduced together into the medium.

  • View full-size image.
  • Figure 5. 

    Regulation of mammalian target of rapamycin (mTOR) expression in bovine mammary gland explants by leptin (L; 0 and 100ng/mL) and prolactin (P; 0 and 1μg/mL) in the presence of insulin (I; 1μg/mL). Western blot (WB) analysis showed that leptin combined with prolactin upregulated the expression of mTOR by approximately 66%. Results are least squares means of the scanning results ± SE (n=3, P<0.05, SEM=0.12). A,BBars with different letters differ (P<0.05).

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Discussion 

In the late 1970s, Phillippy and McCarthy (1979) showed that albumin could be synthesized by the mammary gland in goats. Recent research by our group supports their results by showing that the bovine mammary gland can synthesize albumin de novo and that this synthesis reacts to different physiological conditions (Shamay et al., 2005). The antiapoptotic effect of albumin was demonstrated in cell systems, human endothelial cells (Zoellner et al., 1996), and murine peritoneal macrophages (Koh et al., 1998). Albumin has the ability to bind many fatty acids and small molecules (Berry et al., 2004). Leptin is a hormone secreted mainly by adipose tissue that has many physiological functions (Zhang et al., 1994). Although its biological functions are attributed mainly to body weight regulation, accumulated data suggest that leptin could be involved in the regulation of peripheral tissue metabolism. Previously, we demonstrated that leptin upregulated the effect of prolactin in the bovine mammary gland in vitro (Feuermann et al., 2004). Because leptin upregulated the effect of prolactin on fat synthesis and milk protein expression in mammary gland explants and because albumin is produced by the mammary gland, we asked whether albumin expression might be affected by leptin presence in the incubation medium. It was shown here for the first time that leptin in the presence of prolactin enhanced the expression and secretion of albumin from the bovine mammary gland. It was hypothesized that the presence of albumin played an essential role in cell membrane protection against oxidative and inflammatory agents during concurrent lactation (Ek-Von Mentzer et al., 2001).

Levieux and Ollier (1999) showed that albumin concentration and yield decreased in the early postpartum period. Reist et al. (2003) observed the same phenomenon when they measured leptin concentrations in bovine blood and found a causal relationship between leptin and albumin in lactating dairy cows. Although these latter findings may contradict our hypothesis, we believe that albumin's role lies in the mammary gland maintenance during lactation, because it was demonstrated that albumin was synthesized within the mammary gland (Shamay et al., 2005) and was involved in the local metabolism of the gland. Moreover, leptin alone did not affect the expression of β-lactoglobulin or ß-casein (Feuermann et al., 2004), whereas it did up-regulate the expression of albumin, suggesting that this up-regulation was due to the interleukin properties of leptin. Recent evidence has shown the proinflammatory effect of leptin (Lord, 2006), and we showed that albumin was up-regulated during mastitis in the bovine mammary gland (Shamay et al., 2005). Hence, the current results and the previous results suggest a possible orchestrated linkage between leptin and albumin that is involved in the mammary defense mechanism.

We show here that prolactin up-regulates DNA synthesis in mammary tissue from 2 to 5 mo of lactation and that the addition of leptin induces further up-regulation in a dose-responsive manner. Moreover, apoptosis, as reflected by caspase-3 protein level, was down-regulated by prolactin and leptin.

From the above evidence that points to the apparent regulatory effect of leptin on the action of prolactin during lactogenesis and because leptin up-regulates lactation potential, we expected that the increased protein synthesis would be fulfilled by sufficient supply of amino acids. Therefore, we measured APN expression in the cultured mammary explants. Previously, it was shown that APN, an enzyme that plays an important role in protein digestion and absorption in the small intestine, was expressed in the mammary gland of goats and cows. Aminopeptidase N is one of the peptidases involved in providing amino acids for protein synthesis in the mammary gland and its expression was increased in accordance to mammary gland demands in lactating goats (Mabjeesh et al., 2005). Indeed, in the current study, the greatest APN expression was observed in explants cultured with leptin and prolactin. The finding that APN was up-regulated in explant cultures after stimulation with prolactin and leptin was supported by our previous study in which it was demonstrated that prolactin and leptin interact to up-regulate protein synthesis (Feuermann et al., 2004).

One of the central junctions in the regulation of protein synthesis, DNA synthesis, and differentiation is mTOR (Jankiewicz et al., 2006), a member of the phosphatidylinositol kinase-related protein kinase family that has been found in yeast, fungi, plants, worms, flies, and mammals. It is an essential protein in the regulation of such central cell fates as DNA synthesis, differentiation, and apoptosis. It functions as a sensor for mitogen, energy, and nutrient levels (Fingar and Blenis, 2004). Here we show that mTOR was upregulated when leptin was added to medium containing prolactin. Combining our findings regarding the effect of leptin and prolactin on protein and fat synthesis (Feuermann et al., 2004) and their effects on apoptosis and DNA synthesis (this study), the observed mTOR protein level pattern is not surprising.

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Conclusions 

In this study, we ascertained whether the interaction between leptin and prolactin could be extended to traits other than milk protein expression and fat synthesis, which indicate up-regulation of lactogenesis. The interaction between leptin and prolactin affected protein (namely, albumin) synthesis. Leptin and prolactin affected apoptosis and proliferation, confirming our hypothesis that leptin and prolactin have de novo effects on bovine lactogenesis.

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Supplementary data 

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PII: S0022-0302(08)70965-2

doi:10.3168/jds.2008-0988

Journal of Dairy Science
Volume 91, Issue 11 , Pages 4183-4189, November 2008

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