Recombinant Mouse Osteocalcin Secreted by Lactococcus lactis Promotes Glucagon-Like Peptide-1 Induction in STC-1 Cells
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An osteoblastic protein, osteocalcin (OC), exists in vivo in two forms: carboxylated OC, and uncarboxylated or low-carboxylated OC (ucOC). ucOC acts as a hormone to regulate carbon and energy metabolism. Recent studies demonstrated that ucOC exerts insulinotropic effects, mainly through the glucagon-like peptide 1 (GLP-1) pathway. GLP-1 is an insulinotropic hormone secreted by enteroendocrine L cells in the small intestine. Thus, efficient delivery of ucOC to the small intestine may be a new therapeutic option for metabolic diseases such as diabetes and obesity. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse ucOC. Western blotting showed that the engineered strain (designated NZ-OC) produces and secretes the designed peptide (rOC) in the presence of nisin, an inducer of the recombinant gene. Highly-purified rOC was obtained from the culture supernatants of NZ-OC using immobilized metal affinity chromatography. An in vitro assay showed that purified rOC promotes GLP-1 secretion in a mouse intestinal neuroendocrine cell line, STC-1, in a dose-dependent manner. These results clearly demonstrate that NZ-OC secretes rOC, and that rOC can promote GLP-1 secretion by STC-1 cells. Genetically modified lactic acid bacteria (gmLAB) have been proposed over the last two decades as an effective and low-cost mucosal delivery vehicle for biomedical proteins. NZ-OC may be an attractive tool for the delivery of rOC to trigger GLP-1 secretion in the small intestine to treat diabetes and obesity.
This study was funded by a Grant-in-Aid from the Japan Society for the Promotion of Science Fellows (No. 14J06317) to SS and a grant from the Faculty of Agriculture, Shinshu University, to TSh.
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Conflict of interest
The authors declare that they have no conflict of interest.
- 2.Ferron M, Hinoi E, Karsenty G, Ducy P (2008) Osteocalcin differentially regulates β cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice. Proc Natl Acad Sci USA 105(13):5266–5270. doi: 10.1073/pnas.0711119105 CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, Mee PJ, McKee MD, Jung DY, Zhang Z, Kim JK, Mauvais-Jarvis F, Ducy P, Karsenty G (2007) Endocrine regulation of energy metabolism by the skeleton. Cell 130(3):456–469. doi: 10.1016/j.cell.2007.05.047 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Rishavy MA, Hallgren KW, Yakubenko AV, Zuerner RL, Runge KW, Berkner KL (2005) The vitamin K-dependent carboxylase has been acquired by Leptospira pathogens and shows altered activity that suggests a role other than protein carboxylation. J Biol Chem 280(41):34870–34877. doi: 10.1074/jbc.M504345200 CrossRefPubMedGoogle Scholar
- 15.Samazan F, Rokbi B, Seguin D, Telles F, Gautier V, Richarme G, Chevret D, Varela PF, Velours C, Poquet I (2015) Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis. Microb Cell Fact 14:104. doi: 10.1186/s12934-015-0271-z CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
- 19.Shigemori S, Watanabe T, Kudoh K, Ihara M, Nigar S, Yamamoto Y, Suda Y, Sato T, Kitazawa H, Shimosato T (2015) Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice. Microb Cell Fact 14:189. doi: 10.1186/s12934-015-0378-2 CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Shigemori S, Ihara M, Sato T, Yamamoto Y, Nigar S, Ogita T, Shimosato T (2017) Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis. Appl Microbiol Biotechnol 101(1):341–349. doi: 10.1007/s00253-016-7907-8 CrossRefPubMedGoogle Scholar