Metabolic Effects of a Stabilizing Peptide Fusion Protein of Leptin in Normal Mice

Abstract

Leptin is a protein hormone produced by adipocytes. It is secreted into the blood stream and plays a key role in regulating body energy homeostasis by inhibiting feeding behavior followed by decreased body weight. Because protein aggregation is a major problem in therapeutic proteins, we previously demonstrated that a stabilizing peptide (SP) fusion protein of leptin (SP-leptin) appeared to resist aggregation induced by agitation, freezing/thawing, or heat stress. In this study, we fused mouse leptin with the stabilizing peptide and compared the biological activities of leptin and SP-leptin in vivo using a male C57Bl mouse model and ex vivo using MCF7 breast cancer cell lines. Each group of mice was treated with saline, leptin, and SP-leptin for 20 days and the differences in body weight, food intake, abdominal fat contents, and TG concentration were measured. The SP-leptin appeared to decrease the body weight and food intake in male C57Bl mice more significantly than wild type leptin, and the SP-leptin treated MCF7 cells displayed better cell proliferation than leptin. As a consequence of decreased body weight, the SP-leptin treated mouse group showed decreased abdominal fat contents and low triglyceride (TG) concentration. Moreover, the SP-leptin treated mouse group had fewer lipid droplets in liver and reduced lipid droplet size when analyzed by Oil red O and H & E staining. These results demonstrated that SP-leptin is more effective than wild type leptin in normal mice in lowering their body weight and fat contents in the abdominal region, the serum, and the liver.

Key words

leptin - protein stability - stabilizing peptide - fusion protein - animal experiment - bioefficacy

• References

• 1 Robaczyk M, Smiarowska M, Krzyzanowska-Swiniarska B. The ob gene product (leptin) – a new hormone of adipose tissue. Przegl Lek 1997; 54: 348-352
• 2 Havel PJ. Role of adipose tissue in body-weight regulation: mechanisms regulating leptin production and energy balance. Proc Nutr Soc 2000; 59: 359-371
• 3 Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425-432
• 4 Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995; 269: 543-546
• 5 Wauters M, Considine RV, Yudkin JS, Peiffer F, De Leeuw I, Van Gaal LF. Leptin levels in type 2 diabetes: associations with measures of insulin resistance and insulin secretion. Horm Metab Res 2003; 35: 92-96
• 6 Pardridge WM. Receptor-mediated peptide transport through the blood-brain barrier. Endocr Rev 1986; 7: 314-330
• 7 Cervero A, Dominguez F, Horcajadas JA, Quinonero A, Pellicer A, Simon C. The role of the leptin in reproduction. Curr Opin Obstet Gynecol 2006; 18: 297-303
• 8 Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA 1997; 94: 8878-8883
• 9 Gibson WT, Farooqi IS, Moreau M, DePaoli AM, Lawrence E, O’Rahilly S, Trussell RA. Congenital leptin deficiency due to homozygosity for the Delta133G mutation: report of another case and evaluation of response to four years of leptin therapy. J Clin Endocrinol Metab 2004; 89: 4821-4826
• 10 Fantuzzi G. Leptin: nourishment for the immune system. Eur J Immunol 2006; 36: 3101-3104
• 11 Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995; 269: 540-543
• 12 Cusin I, Sainsbury A, Doyle P, Rohner-Jeanrenaud F, Jeanrenaud B. The ob gene and insulin. A relationship leading to clues to the understanding of obesity. Diabetes 1995; 44: 1467-1470
• 13 Lo KM, Zhang J, Sun Y, Morelli B, Lan Y, Lauder S, Brunkhorst B, Webster G, Hallakou-Bozec S, Doare L, Gillies SD. Engineering a pharmacologically superior form of leptin for the treatment of obesity. Protein Eng Des Sel 2005; 18: 1-10
• 14 Welt CK, Chan JL, Bullen J, Murphy R, Smith P, DePaoli AM, Karalis A, Mantzoros CS. Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med 2004; 351: 987-997
• 15 Mauvais-Jarvis F. Leptin-replacement therapy in lipodystrophy. N Engl J Med 2002; 346 2008; author reply 2009–2010
• 16 Manning MC, Patel K, Borchardt RT. Stability of protein pharmaceuticals. Pharm Res 1989; 6: 903-918
• 17 Cleland JL, Powell MF, Shire SJ. The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carrier Syst 1993; 10: 307-377
• 18 Raso SW, Abel J, Barnes JM, Maloney KM, Pipes G, Treuheit MJ, King J, Brems DN. Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state. Protein Sci 2005; 14: 2246-2257
• 19 Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm 2005; 289: 1-30
• 20 Braun A, Kwee L, Labow MA, Alsenz J. Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice. Pharm Res 1997; 14: 1472-1478
• 21 Maislos M, Bialer M, Mead PM, Robbins DC. Pharmacokinetic model of circulating covalent aggregates of insulin. Diabetes 1988; 37: 1059-1063
• 22 Thornton CA, Ballow M. Safety of intravenous immunoglobulin. Arch Neurol 1993; 50: 135-136
• 23 Ratner RE, Phillips TM, Steiner M. Persistent cutaneous insulin allergy resulting from high-molecular-weight insulin aggregates. Diabetes 1990; 39: 728-733
• 24 Park SM, Jung HY, Chung KC, Rhim H, Park JH, Kim J. Stress-induced aggregation profiles of GST-alpha-synuclein fusion proteins: role of the C-terminal acidic tail of alpha-synuclein in protein thermosolubility and stability. Biochemistry 2002; 41: 4137-4146
• 25 Park SM, Ahn KJ, Jung HY, Park JH, Kim J. Effects of novel peptides derived from the acidic tail of synuclein (ATS) on the aggregation and stability of fusion proteins. Protein Eng Des Sel 2004; 17: 251-260
• 26 Lee EN, Kim YM, Lee HJ, Park SW, Jung HY, Lee JM, Ahn YH, Kim J. Stabilizing peptide fusion for solving the stability and solubility problems of therapeutic proteins. Pharm Res 2005; 22: 1735-1746
• 27 Kim YM, Lee HJ, Lee JE, Kim HY, Kim J. Expression of human interferon alpha-1 with enhanced stability via the tagging system of a stabilizing peptide. Protein Expr Purif 2009; 63: 140-146
• 28 Elinav E, Niv-Spector L, Katz M, Price TO, Ali M, Yacobovitz M, Solomon G, Reicher S, Lynch JL, Halpern Z, Banks WA, Gertler A. Pegylated leptin antagonist is a potent orexigenic agent: preparation and mechanism of activity. Endocrinology 2009; 150: 3083-3091
• 29 Kim J, Chwae YJ, Kim MY, Choi IH, Park JH, Kim SJ. Molecular basis of HLA-C recognition by p58 natural killer cell inhibitory receptors. J Immunol 1997; 159: 3875-3882
• 30 Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LC, Panda AK. Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expr Purif 2000; 18: 182-192
• 31 Liu YF, Wu Q, Xu XM, Ren Y, Yu LN, Quan CS, Li YL. Effects of 17beta-estradiol on proliferation and migration of MCF-7 cell by regulating expression of claudin-6. Zhonghua Bing Li Xue Za Zhi 2010; 39: 44-47
• 32 Dieudonne MN, Machinal-Quelin F, Serazin-Leroy V, Leneveu MC, Pecquery R, Giudicelli Y. Leptin mediates a proliferative response in human MCF7 breast cancer cells. Biochem Biophys Res Commun 2002; 293: 622-628
• 33 Toyoshima Y, Gavrilova O, Yakar S, Jou W, Pack S, Asghar Z, Wheeler MB, LeRoith D. Leptin improves insulin resistance and hyperglycemia in a mouse model of type 2 diabetes. Endocrinology 2005; 146: 4024-4035
• 34 Kim J. Evidence that the precursor protein of non-A beta component of Alzheimer’s disease amyloid (NACP) has an extended structure primarily composed of random-coil. Mol Cells 1997; 7: 78-83
• 35 Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury Jr PT. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 1996; 35: 13709-13715
• 36 Han H, Weinreb PH, Lansbury Jr PT. The core Alzheimer’s peptide NAC forms amyloid fibrils which seed and are seeded by beta-amyloid: is NAC a common trigger or target in neurodegenerative disease?. Chem Biol 1995; 2: 163-169
• 37 Iwai A, Yoshimoto M, Masliah E, Saitoh T. Non-A beta component of Alzheimer’s disease amyloid (NAC) is amyloidogenic. Biochemistry 1995; 34: 10139-10145
• 38 O’Neil JS, Burow ME, Green AE, McLachlan JA, Henson MC. Effects of estrogen on leptin gene promoter activation in MCF-7 breast cancer and JEG-3 choriocarcinoma cells: selective regulation via estrogen receptors alpha and beta. Mol Cell Endocrinol 2001; 176: 67-75
• 39 Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions and new insights. Science 2011; 332: 1519-1523