Recombinant Lactococcus lactis NZ3900 expressing bioactive human FGF21 reduced body weight of Db/Db mice through the activity of brown adipose tissue
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Fibroblast growth factor 21 (FGF21), a metabolism regulator, has an important effect on metabolic diseases, such as obesity and diabetes. It is also expressed in mice, and the murine source has high homology with human FGF21. Recently, it has been extensively studied and has become a potential drug target for the treatment of metabolic diseases. As it is a protein-based hormone, FGF21 cannot be easily and quickly absorbed into the blood through oral administration. Moreover, it has a 0-2 h half-life in vivo, as shown in a previous study, thus its efficacy lasts for a short period of time when used to treat metabolic diseases, limiting its clinical applications. To avoid these limitations, we used Lactococcus lactis, a food-grade bacterium, as the host to express FGF21. It could be used successfully for the expression and long-term effect of FGF21 in vivo. Instead of antibiotic resistance genes, the LacF gene was used as a selection marker in the NZ3900/PNZ8149 expression system, which is safe and could reduce the antibiotic resistance crisis. In this study, we a constructed human FGF21 expressing L. lactis strain and administered it to Db/Db mice by gavage. Compared with the control group, the body weight of mice in the experimental group was significantly reduced, and the overall homeostasis was improved in mice treated with human FGF21. Moreover, the activity of brown adipose tissue was enhanced. These results revealed that oral administration of FGF21 through heterologous expression in L. lactis appears to be an effective approach for its clinical application.
-
Ainsworth, S., Stockdale, S., Bottacini, F., Mahony, J. and van Sinderen, D., 2014. The Lactococcus lactis plasmidome: much learnt, yet still lots to discover. FEMS Microbiology Reviews 38: 1066-1088. https://doi.org/10.1111/1574-6976.12074
-
Alander, M., Satokari, R., Korpela, R., Saxelin, M., Vilpponen-Salmela, T., Mattila-Sandholm, T. and von Wright, A., 1999. Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Applied and Environmental Microbiology 65: 351-354.
'Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption ' () 65 Applied and Environmental Microbiology : 351 -354.
-
Angelin, B., Larsson, T.E. and Rudling, M., 2012. Circulating fibroblast growth factors as metabolic regulators--a critical appraisal. Cell Metabolism 16: 693-705. https://doi.org/10.1016/j.cmet.2012.11.001
-
Bahey-El-Din, M. and Gahan, C.G., 2010. Lactococcus lactis: from the dairy industry to antigen and therapeutic protein delivery. Discovery Medicine 9: 455-461.
'Lactococcus lactis: from the dairy industry to antigen and therapeutic protein delivery ' () 9 Discovery Medicine : 455 -461.
-
Bain, S.C., Feher, M., Russell-Jones, D. and Khunti, K., 2016. Management of type 2 diabetes: the current situation and key opportunities to improve care in the UK. Diabetes, Obesity and Metabolism 18: 1157-1166. https://doi.org/10.1111/dom.12760
-
Baretic, M., 2013. Obesity drug therapy. Minerva Endocrinologica 38: 245-254.
'Obesity drug therapy ' () 38 Minerva Endocrinologica : 245 -254.
-
Barthel, A., Rietzsch, H., Bornstein, S.R. and Schwarz, P., 2011. [Recent advances in type 2 diabetes mellitus]. Deutsche Medizinische Wochenschrift 136: 675-678. https://doi.org/10.1055/s-0031-1274562
-
Benedini, S. and Luzi, L., 2016. Lipodystrophy HIV-related and FGF21: a new marker to follow the progression of lipodystrophy? J Transl Int Med 4: 150-154. https://doi.org/10.1515/jtim-2016-0026
-
Boon, M.R. and van Marken Lichtenbelt, W.D., 2016. Brown adipose tissue: a human perspective. Handbook of Experimental Pharmacology 233: 301-319. https://doi.org/10.1007/164_2015_11
-
Cannon, B. and Nedergaard, J., 2004. Brown adipose tissue: function and physiological significance. Physiological Reviews 84: 277-359. https://doi.org/10.1152/physrev.00015.2003
-
Charteris, W.P., Kelly, P.M., Morelli, L. and Collins, J.K., 1998. Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. Journal of Applied Microbiology 84: 759-768.
'Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract ' () 84 Journal of Applied Microbiology : 759 -768.
-
Coskun, T., Bina, H.A., Schneider, M.A., Dunbar, J.D., Hu, C.C., Chen, Y., Moller, D.E. and Kharitonenkov, A., 2008. Fibroblast growth factor 21 corrects obesity in mice. Endocrinology 149: 6018-6027. https://doi.org/10.1210/en.2008-0816
-
De Ruyter, P.G., Kuipers, O.P. and de Vos, W.M., 1996a. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Applied and Environmental Microbiology 62: 3662-3667.
'Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin ' () 62 Applied and Environmental Microbiology : 3662 -3667.
-
De Ruyter, P.G., Kuipers, O.P. and de Vos, W.M., 1996b. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Applied and Environmental Microbiology 62: 3662-3667.
'Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin ' () 62 Applied and Environmental Microbiology : 3662 -3667.
-
Dejkhamron, P., Menon, R.K. and Sperling, M.A., 2007. Childhood diabetes mellitus: recent advances & future prospects. Indian Journal of Medical Research 125: 231-250.
'Childhood diabetes mellitus: recent advances & future prospects ' () 125 Indian Journal of Medical Research : 231 -250.
-
Dobson, A., Cotter, P.D., Ross, R.P. and Hill, C., 2012. Bacteriocin production: a probiotic trait? Applied and Environmental Microbiology 78: 1-6. https://doi.org/10.1128/AEM.05576-11
-
Fisher, F.M., Estall, J.L., Adams, A.C., Antonellis, P.J., Bina, H.A., Flier, J.S., Kharitonenkov, A., Spiegelman, B.M. and Maratos-Flier, E., 2011. Integrated regulation of hepatic metabolism by fibroblast growth factor 21 (FGF21) in vivo. Endocrinology 152: 2996-3004. https://doi.org/10.1210/en.2011-0281
-
Fisher, F.M. and Maratos-Flier, E., 2016. Understanding the Physiology of FGF21. Annual Review of Physiology 78: 223-241. https://doi.org/10.1146/annurev-physiol-021115-105339
-
Giralt, M., Gavalda-Navarro, A. and Villarroya, F., 2015. Fibroblast growth factor-21, energy balance and obesity. Molecular and Cellular Endocrinology 418 Part 1: 66-73. https://doi.org/10.1016/j.mce.2015.09.018
-
Hanssen, M.J., Broeders, E., Samms, R.J., Vosselman, M.J., Van der Lans, A.A., Cheng, C.C., Adams, A.C., Van Marken Lichtenbelt, W.D. and Schrauwen, P., 2015. Serum FGF21 levels are associated with brown adipose tissue activity in humans. Scientific Reports 5: 10275. https://doi.org/10.1038/srep10275
-
Holmes, D., 2016. Adipose tissue: key role for FGF21 in GLP1-mediated weight loss. Nature Reviews: Endocrinology 12: 688. https://doi.org/10.1038/nrendo.2016.166
-
Hondares, E., Iglesias, R., Giralt, A., Gonzalez, F.J., Giralt, M., Mampel, T. and Villarroya, F., 2011. Thermogenic activation induces FGF21 expression and release in brown adipose tissue. Journal of Biological Chemistry 286: 12983-12990. https://doi.org/10.1074/jbc.M110.215889
-
Huang, J., Ishino, T., Chen, G., Rolzin, P., Osothprarop, T.F., Retting, K., Li, L., Jin, P., Matin, M.J., Huyghe, B., Talukdar, S., Bradshaw, C.W., Palanki, M., Violand, B.N., Woodnutt, G., Lappe, R.W., Ogilvie, K. and Levin, N., 2013. Development of a novel long-acting antidiabetic FGF21 mimetic by targeted conjugation to a scaffold antibody. Journal of Pharmacology and Experimental Therapeutics 346: 270-280. https://doi.org/10.1124/jpet.113.204420
-
Hugenholtz, J. and Smid, E.J., 2002. Nutraceutical production with food-grade microorganisms. Current Opinion in Biotechnology 13: 497-507.
'Nutraceutical production with food-grade microorganisms ' () 13 Current Opinion in Biotechnology : 497 -507.
-
Itoh, N., 2014. FGF21 as a hepatokine, adipokine, and myokine in metabolism and diseases. Frontiers in Endocrinology 5: 107. https://doi.org/10.3389/fendo.2014.00107
-
Jones, B.J. and Bloom, S.R., 2015. The new era of drug therapy for obesity: the evidence and the expectations. Drugs 75: 935-945. https://doi.org/10.1007/s40265-015-0410-1
-
Kharitonenkov, A. and Adams, A.C., 2014. Inventing new medicines: the FGF21 story. Molecular Metabolism 3: 221-229. https://doi.org/10.1016/j.molmet.2013.12.003
-
Kharitonenkov, A., Shiyanova, T.L., Koester, A., Ford, A.M., Micanovic, R., Galbreath, E.J., Sandusky, G.E., Hammond, L.J., Moyers, J.S., Owens, R.A., Gromada, J., Brozinick, J.T., Hawkins, E.D., Wroblewski, V.J., Li, D.S., Mehrbod, F., Jaskunas, S.R. and Shanafelt, A.B., 2005. FGF-21 as a novel metabolic regulator. Journal of Clinical Investigation 115: 1627-1635. https://doi.org/10.1172/JCI23606
-
Kharitonenkov, A., Wroblewski, V.J., Koester, A., Chen, Y.F., Clutinger, C.K., Tigno, X.T., Hansen, B.C., Shanafelt, A.B. and Etgen, G.J., 2007. The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21. Endocrinology 148: 774-781. https://doi.org/10.1210/en.2006-1168
-
Kim, K.H. and Lee, M.S., 2014. FGF21 as a stress hormone: the roles of FGF21 in stress adaptation and the treatment of metabolic diseases. Diabetes & Metabolism Journal 38: 245-251. https://doi.org/10.4093/dmj.2014.38.4.245
-
Kim, K.H. and Lee, M.S., 2015. FGF21 as a mediator of adaptive responses to stress and metabolic benefits of anti-diabetic drugs. Journal of Endocrinology 226: R1-16. https://doi.org/10.1530/JOE-15-0160
-
Li, L.I. and Tang, L., 2015. Multiple roles of fibroblast growth factor 21 in metabolism. Current Pharmaceutical Design 21: 3041-3050.
'Multiple roles of fibroblast growth factor 21 in metabolism ' () 21 Current Pharmaceutical Design : 3041 -3050.
-
Link, A.J. and LaBaer, J., 2011. Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harbour Protocols 2011: 993-994. https://doi.org/10.1101/pdb.prot5651
-
Marakova, E.N. and Bazhan, N.M., 2016. [The role of fibroblast growth factor 21 (Fgf21) in the regulation and correction of carbohydrate and lipid metabolism]. Rossiiskii Fiziologicheskii Zhurnal Imeni I. M. Sechenova 102: 1406-1416.
'[The role of fibroblast growth factor 21 (Fgf21) in the regulation and correction of carbohydrate and lipid metabolism] ' () 102 Sechenova : 1406 -1416.
-
Maratos-Flier, E., 2017. Fatty liver and FGF21 physiology. Experimental Cell Research 360: 2-5. https://doi.org/10.1016/j.yexcr.2017.05.006
-
Mierau, I. and Kleerebezem, M., 2005. 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Applied Microbiology and Biotechnology 68: 705-717. https://doi.org/10.1007/s00253-005-0107-6
-
Mu, J., Pinkstaff, J., Li, Z., Skidmore, L., Li, N., Myler, H., Dallas-Yang, Q., Putnam, A.M., Yao, J., Bussell, S., Wu, M., Norman, T.C., Rodriguez, C.G., Kimmel, B., Metzger, J.M., Manibusan, A., Lee, D., Zaller, D.M., Zhang, B.B., DiMarchi, R.D., Berger, J.P. and Axelrod, D.W., 2012. FGF21 analogs of sustained action enabled by orthogonal biosynthesis demonstrate enhanced antidiabetic pharmacology in rodents. Diabetes 61: 505-512. https://doi.org/10.2337/db11-0838
-
Nakazato, M., 2013. [Current status of medical therapy for obesity and the potential of novel anti-obesity drug development]. Japanese Journal of Clinical Medicine 71: 324-328.
'[Current status of medical therapy for obesity and the potential of novel anti-obesity drug development] ' () 71 Japanese Journal of Clinical Medicine : 324 -328.
-
Ng, D.T. and Sarkar, C.A., 2011. Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000. Biotechnology and Bioengineering 108: 1987-1996. https://doi.org/10.1002/bit.23130
-
Oelkrug, R., Polymeropoulos, E.T. and Jastroch, M., 2015. Brown adipose tissue: physiological function and evolutionary significance. Journal of Comparative Physiology B 185: 587-606. https://doi.org/10.1007/s00360-015-0907-7
-
Olejnik-Schmidt, A., Gronowalska, A. and Schmidt, M., 2013. [The NICE system as a tool for protein expression in Lactococcus lactis]. Postepy Biochemii 59: 322-326.
'[The NICE system as a tool for protein expression in Lactococcus lactis] ' () 59 Postepy Biochemii : 322 -326.
-
Osmanagaoglu, O., Kiran, F. and Ataoglu, H., 2010. Evaluation of in vitro probiotic potential of Pediococcus pentosaceus OZF isolated from human breast milk. Probiotics and Antimicrobial Proteins 2: 162-174. https://doi.org/10.1007/s12602-010-9050-7
-
Owen, B.M., Ding, X., Morgan, D.A., Coate, K.C., Bookout, A.L., Rahmouni, K., Kliewer, S.A. and Mangelsdorf, D.J., 2014. FGF21 acts centrally to induce sympathetic nerve activity, energy expenditure, and weight loss. Cell Metababolism 20: 670-677. https://doi.org/10.1016/j.cmet.2014.07.012
-
Poher, A.L., Altirriba, J., Veyrat-Durebex, C. and Rohner-Jeanrenaud, F., 2015. Brown adipose tissue activity as a target for the treatment of obesity/insulin resistance. Frontiers in Physiology 6: 4. https://doi.org/10.3389/fphys.2015.00004
-
Pontes, D.S., De Azevedo, M.S., Chatel, J.M., Langella, P., Azevedo, V. and Miyoshi, A., 2011. Lactococcus lactis as a live vector: heterologous protein production and DNA delivery systems. Protein Expression and Purification 79: 165-175. https://doi.org/10.1016/j.pep.2011.06.005
-
Pouwels, P.H. and Leer, R.J., 1993. Genetics of lactobacilli: plasmids and gene expression. Antonie Van Leeuwenhoek 64: 85-107.
'Genetics of lactobacilli: plasmids and gene expression ' () 64 Antonie Van Leeuwenhoek : 85 -107.
-
Recinella, L., Leone, S., Ferrante, C., Chiavaroli, A., Di Nisio, C., Martinotti, S., Vacca, M., Brunetti, L. and Orlando, G., 2017. Effects of central fibroblast growth factor 21 (FGF21) in energy balance. Journal of Biological Regulators and Homeostatic Agents 31: 603-613.
'Effects of central fibroblast growth factor 21 (FGF21) in energy balance ' () 31 Journal of Biological Regulators and Homeostatic Agents : 603 -613.
-
Renye, J.A., Jr. and Somkuti, G.A., 2010. Nisin-induced expression of pediocin in dairy lactic acid bacteria. Journal of Applied Microbiology 108: 2142-2151. https://doi.org/10.1111/j.1365-2672.2009.04615.x
-
Saito, M., 2013. Brown adipose tissue as a therapeutic target for human obesity. Obesity Research & Clinical Practice 7: e432-438.
'Brown adipose tissue as a therapeutic target for human obesity ' () 7 Obesity Research & Clinical Practice : e432 -438.
-
Sarruf, D.A., Thaler, J.P., Morton, G.J., German, J., Fischer, J.D., Ogimoto, K. and Schwartz, M.W., 2010. Fibroblast growth factor 21 action in the brain increases energy expenditure and insulin sensitivity in obese rats. Diabetes 59: 1817-1824. https://doi.org/10.2337/db09-1878
-
Schloot, N.C., Roden, M., Bornstein, S.R. and Brendel, M.D., 2011. [Recent advances in the treatment of type 1 diabetes mellitus]. Deutsche Medizinische Wochenschrift 136: 172-175. https://doi.org/10.1055/s-0031-1272502
-
Seo, J.A. and Kim, N.H., 2012. Fibroblast growth factor 21: a novel metabolic regulator. Diabetes and Metabolism Journal 36: 26-28. https://doi.org/10.4093/dmj.2012.36.1.26
-
Smith, R., Duguay, A., Weiszmann, J., Stanislaus, S., Belouski, E., Cai, L., Yie, J., Xu, J., Gupte, J., Wu, X. and Li, Y., 2013. A novel approach to improve the function of FGF21. Biodrugs 27: 159-166. https://doi.org/10.1007/s40259-013-0013-x
-
Stochaj, W.R., Berkelman, T. and Laird, N., 2007. Mass spectrometry-compatible silver staining. Cold Spring Harbour Protocols 2007: pdb prot4742. https://doi.org/10.1101/pdb.prot4742
-
Straub, L. and Wolfrum, C., 2015. FGF21, energy expenditure and weight loss - How much brown fat do you need? Molecular Metabolism 4: 605-609. https://doi.org/10.1016/j.molmet.2015.06.008
-
Van Asseldonk, M., Rutten, G., Oteman, M., Siezen, R.J., de Vos, W.M. and Simons, G., 1990. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 95: 155-160.
'Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp ' () 95 Gene : 155 -160.
-
Veniant, M.M., Komorowski, R., Chen, P., Stanislaus, S., Winters, K., Hager, T., Zhou, L., Wada, R., Hecht, R. and Xu, J., 2012. Long-acting FGF21 has enhanced efficacy in diet-induced obese mice and in obese rhesus monkeys. Endocrinology 153: 4192-4203. https://doi.org/10.1210/en.2012-1211
-
Villarroya, J., Cereijo, R. and Villarroya, F., 2013. An endocrine role for brown adipose tissue? American Journal of Physiology Endocrinology and Metabolism 305: E567-E572. https://doi.org/10.1152/ajpendo.00250.2013
-
Xu, J., Lloyd, D.J., Hale, C., Stanislaus, S., Chen, M., Sivits, G., Vonderfecht, S., Hecht, R., Li, Y.S., Lindberg, R.A., Chen, J.L., Jung, D.Y., Zhang, Z., Ko, H.J., Kim, J.K. and Veniant, M.M., 2009. Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 58: 250-259. https://doi.org/10.2337/db08-0392
-
Yagnik, B., Patel, S., Dave, M., Sharma, D., Padh, H. and Desai, P., 2016. Factors affecting inducible expression of outer membrane protein A (OmpA) of Shigella dysenteriae type-1 in Lactococcus lactis using nisin inducible controlled expression (NICE). Indian Journal of Microbiology 56: 80-87. https://doi.org/10.1007/s12088-015-0556-2
-
Ye, X., Qi, J., Sun, G., Ren, G., Zhu, S., Wu, Y., Yu, D., Zhao, J., Liu, M. and Li, D., 2013. Enhancement of the pharmacological efficacy of FGF-21 by genetic modification and PEGylation. Current Pharmaceutical Biotechnology 14: 1287-1298.
'Enhancement of the pharmacological efficacy of FGF-21 by genetic modification and PEGylation ' () 14 Current Pharmaceutical Biotechnology : 1287 -1298.
-
Zamri, H.F., Shamsudin, M.N., Rahim, R.A. and Neela, V., 2012. Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity. Vaccine 30: 3231-3238. https://doi.org/10.1016/j.vaccine.2012.02.012
-
Zhang, X.J., Duan, G., Zhang, R. and Fan, Q., 2009. Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis nisin-controlled gene expression (NICE) system and experimental study of its immunoreactivity. Current Microbiology 58: 308-314. https://doi.org/10.1007/s00284-008-9349-8
-
Zoka, A., Somogyi, A. and Firneisz, G., 2012. [Type 1 diabetes mellitus: most recent advances in its pathogenesis and treatment]. Orvosi Hetilap 153: 1047-1056. https://doi.org/10.1556/OH.2012.29413
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Recombinant Lactococcus lactis NZ3900 expressing bioactive human FGF21 reduced body weight of Db/Db mice through the activity of brown adipose tissue
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Fibroblast growth factor 21 (FGF21), a metabolism regulator, has an important effect on metabolic diseases, such as obesity and diabetes. It is also expressed in mice, and the murine source has high homology with human FGF21. Recently, it has been extensively studied and has become a potential drug target for the treatment of metabolic diseases. As it is a protein-based hormone, FGF21 cannot be easily and quickly absorbed into the blood through oral administration. Moreover, it has a 0-2 h half-life in vivo, as shown in a previous study, thus its efficacy lasts for a short period of time when used to treat metabolic diseases, limiting its clinical applications. To avoid these limitations, we used Lactococcus lactis, a food-grade bacterium, as the host to express FGF21. It could be used successfully for the expression and long-term effect of FGF21 in vivo. Instead of antibiotic resistance genes, the LacF gene was used as a selection marker in the NZ3900/PNZ8149 expression system, which is safe and could reduce the antibiotic resistance crisis. In this study, we a constructed human FGF21 expressing L. lactis strain and administered it to Db/Db mice by gavage. Compared with the control group, the body weight of mice in the experimental group was significantly reduced, and the overall homeostasis was improved in mice treated with human FGF21. Moreover, the activity of brown adipose tissue was enhanced. These results revealed that oral administration of FGF21 through heterologous expression in L. lactis appears to be an effective approach for its clinical application.
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