- Volume 17 Issue 8
DOI QR Code
Production of Leptin in E. coli and Its Effect on Glucose and Acetate Transport and Expression of Uncoupling Protein-2 Gene in Adipose Tissues of Korean Cattle (Hanwoo)
- Kim, K.S. (Department of Applied Bioscience and Biotechnology, Biotechnology Research Institute, Inst. of Ag. Sci. and Tech. Chonnam National University) ;
- Baik, M.G. (Department of Applied Bioscience and Biotechnology, Biotechnology Research Institute, Inst. of Ag. Sci. and Tech. Chonnam National University)
- Received : 2003.11.27
- Accepted : 2004.05.04
- Published : 2004.08.01
Leptin has a major role in the regulation of food intake and energy homeostasis. In addition, leptin participates in many physiological functions including regulation of lipid metabolism. Bovine recombinant leptin protein was produced in E. coli cells in order to understand function of leptin in the regulation of lipid metabolism. The leptin expression vector was constructed in pGEX-4T-3 vector and transformed into E. coli BL21 cells. Expression of the GST-leptin fusion protein was induced with IPTG. The fusion protein was purified using glutathione sepharose 4B batch method, and the recombinant leptin was eluted after thrombin protease digestion. The effect of leptin on glucose transport was examined in the differentiated adipocytes of 3T3-L1 cells. Leptin had no effect on basal and insulin-stimulated glucose transport in 3T3-L1 cells (p>0.05). Effect of recombinant leptin on glucose and acetate transport was examined in adipose tissues of Korean cattle (Hanwoo). Insulin stimulated glucose transport in both intramuscular and subcutaneous adipose tissues (p<0.05), but leptin did not affect glucose transport in both adipose tissues (p>0.05). Insulin stimulated acetate transport in bovine adipose tissues (p<0.05), but leptin did not affect acetate transport (p>0.05). Northern and RT-PCR analyses showed that mRNA levels of uncoupling protein-2 were increased by leptin treatment in 3T3-L1 cells without statistical difference (p>0.05). In conclusion, bovine recombinant leptin did not affect glucose and acetate transport in both 3T3-L1 adipocytes and bovine adipose tissues, while it stimulates UCP-2 mRNA expression in 3T3-L1 cells.
Supported by : Ministry of Agriculture-Special
- Bai, Y. L., S. Y. Zhang, K. S. Kim, J. K. Lee and K. H. Kim. 1996. Obese gene expression alters the ability of 30A5 preadipocytes to respond to lipogenic hormones. J. Biol. Chem. 271:13939-13942.
- Berti, L., M. Kellerer, E. Capp and H. U. Haring. 1997. Leptin stimulates glucose transport and glycogen synthesis in C2C12 myotubes: Evidence for a PI3-kinase mediated effect. Diabetologia 40:606-609.
- Ceddia, R. B., W. N. William, F. B. Lima, P. Flandin, R. Curi and J. P. Giacobino. 2000. Leptin stimulates uncoupling protein-2 mRNA expression and Krebs cycle activity and inhibits lipid synthesis in isolated rat white adipocytes. Eur. J. Biochem. 267:5952-5958.
- Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate phenol-chloroform extraction. Anal. Biochem. 162:156-159.
- C. Levi-Meyrueis, F. Bouillaud, M. F. Seldin, R. S. Surwit, D. Ricquier and C. H. Warden. 1997. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat. Genet. 15(3):269-272.
- Flier, J. S. and E. Maratos-Flier. 1998. Obesity and the hypothalamus: Novel peptides for new pathways. Cell 92:437-440.
- Fruhbeck, G., J. Gomez-Ambrosi, F. J. Muruzabal and M. A. Burrell. 2001. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am. J. Physiol. Endocrinol. Metab. 280(6):E827-47.
- Halaas, J. L., K. S. Gajiwala, M. Maffei, S. L. Cohen, B. T. Chait, D. Rabinowitz, R. L. Lallone, S. K. Burley and J. M. Friedman. 1995. Weight-reducing effects of the plasma-protein encoded by the obese gene. Science 269:543-546.
- Houseknecht, K. L. and C. P. Portocarrero. 1998. Leptin and its receptors: Regulators of Whole-body energy homeostasis. Domestic Animal Endocrinology 15:457-475.
- Kim, K.-H. and Y. S. Moon. 2003. Molecular cloning of adipose tissue-specific genes by cDNA microarray. Asian-Aust. J. Anim. Sci. 16:1837-1841.
- Kim, H., C. Yountae, K. Chung, K. Kim, Y. Choi and M. Baik. 2000. Differential response of obese gene expression from fasting in bovine adipose tissues. Biosci. Biotechnol. Biochem. 64:2240-2242.
- Lee, G. H., R. Proenca, J. M. Montez, K. M. Carroll, J. G. Darvishzadeh, J. I. Lee and J. M. Friedman. 1996. Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632-635.
- May, S. G., J. W. Savell, D. K. Lunt, J. J. Wilson, J. C. Laurenz and S. B. Smith. 1994. Evidence for preadipocyte proliferation during culture of subcutaneous and intramuscular adipose tissues from Angus and Wagyu crossbred steers. J. Anim. Sci. 72:3110-3117.
- Mueller, W. M., F. M. Gregoire, K. L. Stanhope, C. V. Mobbs, T. M. Mizuno, C. H. Warden, J. S. Stern and P. J. Havel. 1998. Evidence that glucose metabolism regulates leptin secretion from cultured rat adipocytes. Endocrinology 139:551-558.
- Muller, G., J. Ertl, M. Gerl and G. Preibisch. 1997. Leptin impairs metabolic actions of insulin in isolated rat adipocytes. J. Biol. Chem. 272:10585-10593.
- Muoio, D. M., G. L. Dohn, F. T. Fiedorek, E. B. Tapscott and R. A. Coleman. 1997. Leptin directly alters lipid partitioning in skeletal muscle. Diabetes 46:1360-1363.
- Pelleymounter, M. A., M. J. Callen, M. B. Baker, R. Hecht, D. Winters, T. Boone and F. Collins. 1995. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540-543.
- Pinkney, J. H., O. Boss, G. A. Bray, K. Bulmer, S. W. Coppack and V. Mohamed-Ali. 2000. Physiological relationships of uncoupling protein-2 gene expression in human adipose tissue in vivo. J. Clin. Endocrinol. Metab. 85:2312-2317.
- Ranganathan, S., T. P. Ciaraldi, R. R. Henry, S. Mudaliar and P. A. Kern. 1998. Lack of effect of leptin on glucose transport, lipoprotein lipase, and insulin action in adipose and muscle cells. Endocrinology 139:2509-2513.
- SAS. 1998. SAS/STAT. User’s Guide (Release 6.03 Ed). SAS Inst., Inc., Cary, NC.
- Saladin, R., P. De Vos, M. Guerre-Millo, A. Leturque, J. Girard, B. Staels and J. Auwerx. 1995. Transient increase in obese gene expression after food intake or insulin administration. Nature 377:527-529.
- Sivitz, W. I., B. D. Fink, D. A. Morgan, J. M. Fox, P. A. Donohoue and W. G. Haynes. 1999. Sympathetic inhibition, leptin, and uncoupling protein subtype expression in normal fasting rats. Am. J. Physiol. 277:E668-E677.
- Takahashi, Y., Y. Okimura, I. Mizuno, T. Takahashi, H. Kaji, T. Uchiyama, H. Abe and K. Chihara. 1996. Leptin induces tyrosine phosphorylation of cellular proteins including STAT-1 in human renal adenocarcinoma cells. Biohem. Biophys. Res. Commun. 228:859-864.
- Tellam, R. L. 1996. Bos taurus leptin (obese) gene, complete CDs. Genbank accession #BTU50365.
- Zhang, Y., R. Proenca, M. Maffei, M. Barone, L. Leopold and J. M. Friedman. 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372:425-432.
- Zhou, Y. T., M. Shimabukuro, K. Koyama, Y. Lee, M. Y. Wang, F. Trieu, C. B. Newgard and R. H. Unger. 1997. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proc. Natl. Acad. Sci.USA. 94:6386-6390.