Abstract
Lactoferrin (LF) is a multifunctional protein in mammalian milk. We previously reported that enteric-coated bovine LF reduced the visceral fat in a double-blind clinical study. We further demonstrated that bovine LF (bLF) inhibited adipogenesis and promoted lipolysis in white adipocytes, but the effect of bLF on brown adipocytes has not been clarified. In this study, we investigated the effects of bLF on energy expenditure and cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway using human reprogrammed brown adipocytes generated by gene transduction. bLF at concentrations of ≥ 100 μg/mL significantly increased uncoupling protein 1 (UCP1) mRNA levels, with the maximum value observed 4 h after bLF addition. At the same time point, bLF stimulation also significantly increased oxygen consumption. Signaling pathway analysis revealed rapid increases of intracellular cAMP and cAMP response element-binding protein (CREB) phosphorylation levels beginning 5 min after bLF addition. The mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were also significantly increased after 1 h of bLF stimulation. H-89, a specific PKA inhibitor, abrogated bLF-induced UCP1 gene expression. Moreover, receptor-associated protein (Rap), an antagonist of low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced bLF-induced UCP1 gene expression in a dose-dependent manner. These results suggest that bLF promotes UCP1 gene expression in brown adipocytes through the cAMP-PKA signaling pathway via the LRP1 receptor, leading to increased energy expenditure.
Similar content being viewed by others
References
Abe Y, Rozqie R, Matsumura Y, Kawamura T, Nakaki R, Tsurutani Y, Tanimura-Inagaki K, Shiono A, Magoori K, Nakamura K, Ogi S (2015) JMJD1A is a signal-sensing scaffold that regulates acute chromatin dynamics via SWI/SNF association for thermogenesis. Nat Commun 6:7052. https://doi.org/10.1038/ncomms8052
Baar K (2014) Nutrition and the adaptation to endurance training. Sports Med 44:S5–S12. https://doi.org/10.1007/s40279-014-0146-1
Cannon B, Nedergaard J (2004) Brown adipose tissue: function and physiological significance. Physiol Rev 84:277–359. https://doi.org/10.1152/physrev.00015.2003
Collins S, Yehuda-Shnaidman E, Wang H (2010) Positive and negative control of Ucp1 gene transcription and the role of beta-adrenergic signaling networks. Int J Obes 34:S28–S33. https://doi.org/10.1038/ijo.2010.180
Duvnjak L, Duvnjak M (2009) The metabolic syndrome—an ongoing story. J Physiol Pharmacol 60:19–24
Fenzl A, Kiefer FW (2014) Brown adipose tissue and thermogenesis. Horm Mol Biol Clin Investig 19:25–37. https://doi.org/10.1515/hmbci-2014-0022
Fischer R, Debbabi H, Blais A, Dubarry M, Rautureau M, Boyaka PN, Tome D (2007) Uptake of ingested bovine lactoferrin and its accumulation in adult mouse tissues. Int Immunopharmacol 7:1387–1393. https://doi.org/10.1016/j.intimp.2007.05.019
Fujiwara K, Mori K, Kaneko YS, Nakashima A, Nagasaka A, Itoh M, Ota A (2004) Tetrahydrobiopterin biosynthesis in white and brown adipose tissues is enhanced following intraperitoneal administration of bacterial lipopolysaccharide. Biochem Biophys Acta 1670:181–198. https://doi.org/10.1016/j.bbagen.2003.12.004
Goretzki L, Mueller BM (1998) Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein. Biochem J 336:381–386
Harmsen MC, Swart PJ, de Bethune MP, Pauwels R, De Clercq E, The TH, Meijer DK (1995) Antiviral effects of plasma and milk proteins: lactoferrin shows potent activity against both human immunodeficiency virus and human cytomegalovirus replication in vitro. J Infect Dis 172:380–388
Herz J, Strickland DK (2001) LRP: a multifunctional scavenger and signaling receptor. J Clin Investig 108:779–784. https://doi.org/10.1172/JCI13992
Hofmann SM, Zhou L, Perez-Tilve D, Greer T, Grant E, Wancata L, Thomas A, Pfluger PT, Basford JE, Gilham D, Herz J (2007) Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice. J Clin Investig 117:3271–3282. https://doi.org/10.1172/JCI31929
Ikoma-Seki K, Nakamura K, Morishita S, Ono T, Sugiyama K, Nishino H, Hirano H, Murakoshi M (2015) Role of LRP1 and ERK and cAMP signaling pathways in lactoferrin-induced lipolysis in mature rat adipocytes. PLoS ONE 10:e0141378. https://doi.org/10.1371/journal.pone.0141378
Jiang R, Lopez V, Kelleher SL, Lonnerdal B (2011) Apo- and holo-lactoferrin are both internalized by lactoferrin receptor via clathrin-mediated endocytosis but differentially affect ERK-signaling and cell proliferation in Caco-2 cells. J Cell Physiol 226:3022–3031. https://doi.org/10.1002/jcp.22650
Kishida T, Ejima A, Yamamoto K, Tanaka S, Yamamoto T, Mazda O (2015) Reprogrammed functional brown adipocytes ameliorate insulin resistance and dyslipidemia in diet-induced obesity and type 2 diabetes. Stem Cell Rep 5:569–581. https://doi.org/10.1016/j.stemcr.2015.08.007
Kozu T, Iinuma G, Ohashi Y, Saito Y, Akasu T, Saito D, Alexander DB, Iigo M, Kakizoe T, Tsuda H (2009) Effect of orally administered bovine lactoferrin on the growth of adenomatous colorectal polyps in a randomized, placebo-controlled clinical trial. Cancer Prev Res 2:975–983. https://doi.org/10.1158/1940-6207.CAPR-08-0208
Moreno-Navarrete JM, Ortega FJ, Ricart W, Fernandez-Real JM (2009) Lactoferrin increases (172Thr)AMPK phosphorylation and insulin-induced (p473Ser)AKT while impairing adipocyte differentiation. Int J Obes 33:991–1000. https://doi.org/10.1038/ijo.2009.143
Morishita S, Ono T, Fujisaki C, Ishihara Y, Murakoshi M, Kato H, Hosokawa M, Miyashita K, Sugiyama K, Nishino H (2013) Bovine lactoferrin reduces visceral fat and liver triglycerides in ICR mice. J Oleo Sci 62:97–103
Mota M, Panus C, Mota E, Lichiardopol C, Vladu D, Toma E (2004) The metabolic syndrome—a multifaced disease. Rom J Intern Med 42:247–255
Ono T, Murakoshi M, Suzuki N, Iida N, Ohdera M, Iigo M, Yoshida T, Sugiyama K, Nishino H (2010) Potent anti-obesity effect of enteric-coated lactoferrin: decrease in visceral fat accumulation in Japanese men and women with abdominal obesity after 8-week administration of enteric-coated lactoferrin tablets. Br J Nutr 104:1688–1695. https://doi.org/10.1017/S0007114510002734
Ono T, Morishita S, Fujisaki C, Ohdera M, Murakoshi M, Iida N, Kato H, Miyashita K, Iigo M, Yoshida T, Sugiyama K (2011) Effects of pepsin and trypsin on the anti-adipogenic action of lactoferrin against pre-adipocytes derived from rat mesenteric fat. Br J Nutr 105:200–211. https://doi.org/10.1017/S0007114510003259
Ono T, Fujisaki C, Ishihara Y, Ikoma K, Morishita S, Murakoshi M, Sugiyama K, Kato H, Miyashita K, Yoshida T, Nishino H (2013) Potent lipolytic activity of lactoferrin in mature adipocytes. Biosci Biotechnol Biochem 77:566–571. https://doi.org/10.1271/bbb.120817
Richard D, Carpentier AC, Dore G, Ouellet V, Picard F (2010) Determinants of brown adipocyte development and thermogenesis. Int J Obes 34:S59–S66. https://doi.org/10.1038/ijo.2010.241
Saito M (2013) Brown adipose tissue as a regulator of energy expenditure and body fat in humans. Diabetes Metab 37:22–29. https://doi.org/10.4093/dmj.2013.37.1.22
Saito M, Okamatsu-Ogura Y, Matsushita M, Watanabe K, Yoneshiro T, Nio-Kobayashi J, Iwanaga T, Miyagawa M, Kameya T, Nakada K, Kawai Y (2009) High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity. Diabetes 58:1526–1531. https://doi.org/10.2337/db09-0530
Shoji H, Oguchi S, Shinohara K, Shimizu T, Yamashiro Y (2007) Effects of iron-unsaturated human lactoferrin on hydrogen peroxide-induced oxidative damage in intestinal epithelial cells. Pediatr Res 61:89–92. https://doi.org/10.1203/01.pdr.0000250198.22735.20
Suzuki YA, Lopez V, Lonnerdal B (2005) Mammalian lactoferrin receptors: structure and function. Cell Mol Life Sci 62:2560–2575. https://doi.org/10.1007/s00018-005-5371-1
Takayama Y, Takahashi H, Mizumachi K, Takezawa T (2003) Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts. J Biol Chem 278:22112–22118. https://doi.org/10.1074/jbc.M300894200
Takeuchi T, Shimizu H, Ando K, Harada E (2004) Bovine lactoferrin reduces plasma triacylglycerol and NEFA accompanied by decreased hepatic cholesterol and triacylglycerol contents in rodents. Br J Nutr 91:533–538. https://doi.org/10.1079/BJN20041090
Tomita M, Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K (1991) Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J Dairy Sci 74:4137–4142. https://doi.org/10.3168/jds.S0022-0302(91)78608-6
van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, Bouvy ND, Schrauwen P, Teule GJ (2009) Cold-activated brown adipose tissue in healthy men. N Engl J Med 360:1500–1508. https://doi.org/10.1056/NEJMoa0808718
Vash B, Phung N, Zein S, DeCamp D (1998) Three complement-type repeats of the low-density lipoprotein receptor-related protein define a common binding site for RAP, PAI-1, and lactoferrin. Blood 92:3277–3285
Vogel HJ (2012) Lactoferrin, a bird’s eye view. Biochem Cell Biol 90:233–244. https://doi.org/10.1139/o2012-016
Yagi M, Suzuki N, Takayama T, Arisue M, Kodama T, Yoda Y, Numasaki H, Otsuka K, Ito K (2008) Lactoferrin suppress the adipogenic differentiation of MC3T3-G2/PA6 cells. J Oral Sci 50:419–425
Yoneshiro T, Aita S, Matsushita M, Okamatsu-Ogura Y, Kameya T, Kawai Y, Miyagawa M, Tsujisaki M, Saito M (2011) Age-related decrease in cold-activated brown adipose tissue and accumulation of body fat in healthy humans. Obesity 19:1755–1760. https://doi.org/10.1038/oby.2011.125
Yoneshiro T, Aita S, Matsushita M, Kayahara T, Kameya T, Kawai Y, Iwanaga T, Saito M (2013) Recruited brown adipose tissue as an antiobesity agent in humans. J Clin Investig 123:3404–3408. https://doi.org/10.1172/JCI67803
Ziere GJ, van Dijk MC, Bijsterbosch MK, van Berkel TJ (1992) Lactoferrin uptake by the rat liver. Characterization of the recognition site and effect of selective modification of arginine residues. J Biol Chem 267:11229–11235
Zimecki M, Wlaszczyk A, Cheneau P, Brunel AS, Mazurier J, Spik G, Kubler A (1998) Immunoregulatory effects of a nutritional preparation containing bovine lactoferrin taken orally by healthy individuals. Arch Immunol Ther Exp 46:231–240
Acknowledgements
This research was funded by the Lion Corporation, and all experiments were conducted by researchers hired by the company.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Rights and permissions
About this article
Cite this article
Nakamura, K., Kishida, T., Ejima, A. et al. Bovine lactoferrin promotes energy expenditure via the cAMP-PKA signaling pathway in human reprogrammed brown adipocytes. Biometals 31, 415–424 (2018). https://doi.org/10.1007/s10534-018-0103-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10534-018-0103-9