- Email: [email protected]
Effects of leptin gene expression in mice in vivo by electroporation and hydrodynamics-based gene delivery
BBRC Biochemical and Biophysical Research Communications 307 (2003) 440–445 www.elsevier.com/locate/ybbrc
Effects of leptin gene expression in mice in vivo by electroporation and hydrodynamics-based gene delivery Lan Xiang,a Atsushi Murai,a Kunio Sugahara,b Akihiro Yasui,c and Tatsuo Muramatsua,* a
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan b Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan c Department of Surgery, Aichi-Saiseikai Hospital, Nagoya 451-0052, Japan Received 9 June 2003
Abstract In vivo electroporation and hydrodynamics-based gene delivery were utilized to test the effect of leptin gene transfer on food intake, and body and fat weights of mice. Gene transfer of pVRmob by electroporation caused a significant reduction in body weight compared with the control counterpart (p < 0:05), although a lesser effect was found in food intake, and the weights of interscapular brown and epididymal fat by electroporation. As might be expected, the hydrodynamics-based transfection method significantly reduced body weight over 1 week post-transfection (p < 0:05). Furthermore, epididymal fat was decreased by 50% at 1 week after gene transfer (p < 0:001). These results suggest that both electroporation and hydrodynamics-based gene delivery may be effective approaches for systemic delivery of recombinant leptin to the central nervous system, and that the efficiency of gene transfer in hydrodynamics-based gene delivery was markedly higher than that in electroporation at least within the first week after transfection. Ó 2003 Elsevier Inc. All rights reserved. Keywords: In vivo electroporation; Hydrodynamics-based gene delivery; Leptin expression vector; Food intake; Body weight
Leptin, product of the obese (ob) gene, functions to regulate food intake and energy expenditure as a hormone synthesized in adipose tissue through targeting the receptor in hypothalamus [1–3]. To a certain extent, placental, gastric, and neonatal brown adipocytes can also synthesize and secrete this hormone [4–6]. The major function of leptin produced in these tissues is to control meal size. Administration of recombinant leptin protein per se in ob/ob mice is known to result in reduced body weight and food intake, and increased metabolic rate [7–10]. However, since exogenous leptin administration would diminish these hormonal effects within a short period, daily injection of the hormone is required. Instead of administrating hormone per se, in vivo gene transfer not only mimicked but also prolonged those exogenous hormonal effects [11–16]. Moreover, such in vivo gene transfer methodology is expected to *
Corresponding author. Fax: +81-52-789-4077. E-mail address: [email protected] (T. Muramatsu). 0006-291X/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01172-0
bring about similar effects with remarkably low cost. Among in vivo gene transfer means, virus vector methods would impose an increased possibility of biohazard and result in diminished effects after repeated use due probably to immunogenicity evoked in host animals and humans. In this respect, physical in vivo methods are considered safer and have an advantage over the virus vector ones, although no report on leptin gene transfer has been published so far. In the present study, in vivo electroporation and hydrodynamics-based gene delivery were utilized to transfer leptin genes, and the effects on food intake, growth, and energy metabolism in mice were investigated.
Materials and methods Plasmid construction. The expression vector, pVRmob, was constructed by substituting the mouse leptin gene excised from pBluescript-mob (kindly provided by Dr. J.M. Friedman, Rockefeller
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445 University, USA) for the mouse erythropoietin gene in pVRmEPO (kindly provided by Dr. J.M. Leiden, Chicago University, USA) through restriction and ligation reactions. Experimental animals. Male ICR strain mice at 6–8 weeks of age were used as experimental animals. They were maintained in a clean room at 23 1 °C with a 12L/12D photoperiodic cycle, fed a commercial diet (MR stock, Nihon Nosan, Yokohama, Japan) ad libitum, and freely received water during the experimental period. The mice were cared for under Guideline of Animal Experimentation, laid down by the Committee of Experimental Animal Care, Nagoya University. Gene electroporation. The mice were anesthetized by injecting with pentobarbital intraperitoneally at 25 mg/kg body weight. Two incisions approximately 2 cm long were made in the skin around the leg muscle, M. gastrocnemius, of left and right legs. The two leg muscles were exposed and 500 lg plasmids (pVR empty vector or pVRmob expression vector) in combination with 20 lg pEGFP-C1 (Clontech, California, CA) were injected with a syringe. Square electric pulses were applied six times with an electroporator (CUY21, NEPA Gene, Ichikawa, Japan) by using a pair of pinecette-type electrodes at 25 V with the loading period of 100 ms/pulse. Then, the skin was stitched with no. 3 silk suture. After electroporation, the mice were maintained for up to the subsequent 4 weeks. Food intake and body weight were recorded daily for the entire experimental period. At designated time points during the experiment, blood was taken in a heparinized hematocrit tube for analysis of plasma leptin concentration. At the end of the experiment, treated mice were killed by neck dislocation and leg muscle was removed for analyses of GFP expression, leptin mRNA by RT-PCR, and leptin by Western blotting. GFP expression in the leg muscle was observed with a fluorescent microscope (SZX-RFL2, Olympus, Tokyo, Japan). Epididymal white fat and interscapular brown fat were removed quickly and weighed. Hydrodynamics-based gene delivery. Injection of the plasmid was performed as described by Liu et al. [17]. Briefly, the male mice were anesthetized by injecting with pentobarbital intraperitoneally at 25 mg/ kg body weight. Then, 12.5 lg DNA (pVR empty vector or pVRmob expression vector) in lactated Ringer’s solution (0.1 ml/g body weight) was injected into the tail vein using a syringe with a 27-gauge needle within 10 s. After hydrodynamics-based gene delivery, the mice were maintained for up to the subsequent 2 weeks. Food intake and body weight were recorded daily for the entire experimental period. At designated time points during the experiment, blood was taken as in the electroporation experiment for analysis of plasma leptin concentration. At the end of the experiment, treated mice were killed by neck dislocation and liver was removed for analysis of GFP expression, which was determined with a fluorescent microscope (SZX-RFL2, Olympus, Tokyo, Japan). Epididymal white fat and interscapular brown fat were removed quickly and weighed as in the electroporation experiment. RT-PCR assay. Approximately 50 mg electroporation-treated muscle samples of the transfected area were taken at 3 days after gene transfer. Total RNA was isolated using the Trizol reagent procedure (Life Technologies, Rockville, MD) and RNA content was quantitated spectrophotometrically at 260 nm. RT-PCR was done with a commercially available kit (Takara RNA PCR kit (AMV), Otsu, Japan) to reverse transcribe 1 lg total RNA isolated from the muscle sample using an oligo(dT) adaptor primer, with Perkin–Elmer DNA Thermal Cycler (Takara, Otsu, Japan) and Taq polymerase (Promega, Madison, WI). The mouse leptin-specific primers used for the PCR were as follows: sense, 50 -GAC CCC TGT GTC GGT TCC TGT-30 ; antisense 50 -GCT TCT GCA GGC CAC TGG TCT-30 . Western blot analysis. Approximately 0.2 g electroporation-treated muscle samples were taken at 3 days after gene transfer and placed in a 5 ml centrifuge tube to which 2 ml of lysis buffer (500 mM Tris–HCl, 10% SDS, 50% glycerol, and 500 mM DTT, pH adjusted to 6.8) was added. After homogenization and sonication, they were centrifuged at 12,000g at 4 °C for 3 min and the supernatant was transferred to a new tube. Subsequently, protein concentration was measured by Lowry
441
method with a spectrophotometer at 550 nm. The sample protein at 100 lg was run on a 15% SDS–PAGE gel. The proteins were transferred to a PVDF membrane and immunoblotted with a leptin antibody (Sigma, St. Louis, MO, USA), followed by incubation with the second antibody, anti-rabbit IgG conjugated with horseradish peroxidase (Promega, Madison, WI). Chemiluminescence was measured with a Light-capture (AE-6955, ATTO, Tokyo, Japan). Enzyme-linked immunosorbent assay. The plasma samples at 2 and 4 weeks after gene transfer by electroporation, and at 2, 4, 6, 10, and 14 days after gene transfer by hydrodynamics-based gene delivery were obtained by centrifuging the blood at 6000g at room temperature for 5 min and stored at )20 °C until analysis. The plasma samples were assayed for leptin concentration using a commercially available ELISA kit (mouse leptin ELISA kit, Morinaga, Yokohama, Japan) according to the manufacturer’s protocol. Statistical analysis. Statistical treatment on the data was done by a one-way analysis of variance using a commercially available statistical package, SAS (SAS Institute, Cary, NC), and the significance of difference between means was examined by a protected least significant difference method. Results were expressed as means SEM. A value of P < 0:05 was considered significant.
Results GFP expression in animals after electroporation and hydrodynamics-based gene delivery GFP expression in leg muscle and liver after leptin gene transfer by electroporation and hydrodynamicsbased gene delivery, respectively, is presented in Fig. 1. Clear GFP-specific fluorescence signals were detected along the muscle fibers by electroporation and hepatocytes by hydrodynamics-based gene delivery. This implied that the simultaneously transferred pVRmob was successfully expressed in both the muscle and liver. Detection of leptin mRNA and leptin in leg muscle The results of RT-PCR and Western blot from the electroporation-treated muscle are shown in Figs. 2A and B, respectively. The specific bands for the leptin mRNA and leptin protein of 372 bp and 16 kDa size, respectively, were observed. These results suggested that leptin mRNA and leptin protein were produced adequately in leg muscle after gene transfer in vivo by electroporation. Effects of in vivo electroporation on plasma leptin concentration, food intake, and body weight Fig. 3 displays the values for plasma leptin concentration after gene transfer by electroporation. At both 2 and 4 weeks, the leptin gene transfer group had higher plasma leptin concentrations than did the control counterpart and significant difference was detected at 2 weeks (p < 0:05). There was a significant increase both in the control and pVRmob groups from 2 to 4 weeks after gene transfer (p < 0:05). This age-dependent
442
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445
Fig. 1. GFP gene expression in the mouse leg muscle after in vivo gene transfer by electroporation (A) and in the liver by hydrodynamics-based gene delivery (B). The GFP expression plasmid, pEGFP-C1, was co-transferred at a dose of 20 lg per animal in combination with pVRmob. GFP-specific fluorescence signals were detected with a fluorescent microscope at 7 days after in vivo gene electroporation in muscle (A) and 1 day after hydrodynamics-based gene delivery (B). The arrows show fluorescent-positive myotubes (A) or liver cells (B). Scale is given by a horizontal bar.
Fig. 2. Analysis of pVRmob transcript and the resultant protein products. (A) RT-PCR amplified leptin mRNA in the mouse leg muscle to which pVRmob was transferred in vivo by electroporation at a dose of 500 lg per animal. Lanes: M, 100 bp marker; 1 and 2, control muscle; 3–5, pVRmob transferred muscle. (B) Western blotting of leptin protein in the mouse leg muscle after gene transfer. Lanes: 1, 2, control muscle; 3–5, pVRmob transferred muscle.
observed at 4, 5, and 7 days after in vivo gene transfer in the pVRmob group compared with the control counterpart (p < 0:05). Daily food intake was not significantly different between the two treatment groups. The weights of epididymal, white fat, interscapular brown fat, and liver were little affected by the leptin gene transfer (p > 0:05, data not shown). Effects of hydrodynamics-based gene delivery on plasma leptin concentration, food intake, body weight, and adipose tissue weight
Fig. 3. Changes in plasma leptin concentration after leptin gene transfer in vivo by electroporation at a dose of 500 lg per animal. Vertical bars represent SEM of five mice. *Significantly different at p < 0:05; ns, not significantly different (p > 0:05).
increase may probably be brought about by gross increases in adipose weight due to aging, leading to increased production of leptin in the plasma. Fig. 4 gives the values for body weight and food intake after gene transfer by electroporation. Body weight tended to be decreased and significant difference was
The leptin concentration in plasma after in vivo gene transfer by hydrodynamics-based gene delivery is given in Fig. 5. At 2 days post-transfection, a remarkable increase was detected by a factor of 40 in the leptin gene transferred group compared with the control group, although leptin concentration was sharply decreased afterwards. Significant differences in plasma leptin were detected in the pVRmob group compared with the control counterpart at 2 days (p < 0:001), and 4 and 10 days (p < 0:05). Fig. 6 presents the values for food intake and body weight after gene transfer by hydrodynamics-based gene delivery. Body weight was significantly reduced at 2 days
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445
443
Fig. 4. Changes in body weight and food intake of mice after transferring pVRmob gene in vivo by electroporation at a dose of 500 lg per animal. Each point represents means SEM of five mice. *Significantly different from the control at the same time point at p < 0:05 (*).
and the lowered body weight was maintained for the first 7 days (p < 0:05). The significant reduction in food intake was detected only at 2 days after gene transfer (p < 0:05) and the food intake was quickly restored to the normal level. Fig. 7 evaluates the values for epididymal white fat and interscapular brown fat. These weights tended to be reduced at 1 week and the reduction in epididymal white fat weight reached statistical significance in the treated group compared with the control counterpart (p < 0:001).
Discussion
Fig. 5. Changes in plasma leptin concentration after leptin gene transfer in vivo by hydrodynamics-based gene delivery at a dose of 12.5 lg per animal. Each point represents means SEM of three mice. *, ** Significantly different from the control at the same time point at p < 0:05 (*) and p < 0:001 (**).
In the present study, to monitor whether a foreign gene could be transferred in vivo by electroporation and hydrodynamics-based gene delivery, a GFP expression plasmid, pEGFP-C1, was transferred. The results of strong GFP expression indicated that in vivo gene transfer was successfully completed. RT-PCR
Fig. 6. Changes in body weight and food intake of mice after transferring pVRmob gene in vivo by hydrodynamics-based gene delivery at a dose of 12.5 lg per animal. Each point represents means SEM of five mice. * Significantly different from the control at the same time point at p < 0:05 (*); ns, not significantly different (p > 0:05).
444
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445
In conclusion, the present study provided the first direct evidence of successful leptin gene transfer in mice in vivo by electroporation and hydrodynamics-based gene delivery. These nonviral gene transfer methods would facilitate the better understanding of regulatory mechanisms of leptin on food intake and energy metabolism in vivo.
Acknowledgments We gratefully acknowledge Dr. J.M. Friedman, Rockefeller University, and Dr. J.M. Leiden, Chicago University, USA, for gifts of pBluescript-mob and pVRmEPO plasmids, respectively. This work was in part supported by a Grant-in-Aid for Scientific Research (B) (2) (No. 15380192) from the Japan Society for the Promotion of Science. Fig. 7. Weights of epdidymal white fat and interscapular brown fat of mice to which pVRmob was transferred in vivo by hydrodynamicsbased gene delivery at a dose of 12.5 lg per animal. The adipose tissue weights were measured at 1 week after gene transfer. Vertical bars represent SEM of five mice. ** Significantly different at p < 0:001; ns, not significantly different (p > 0:05).
and Western blotting analyses demonstrated that the simultaneously transfected leptin gene was adequately transcribed and translated. Effects of leptin gene transfer on reduced food intake and decreased body weight were also observed, which were in good agreement with those of leptin administration in the previous studies [9–13]. However, the extent of reduction in body weight was smaller by electroporation (3.5%) than by hydrodynamics-based transfection (6.3%). Significant reductions in adipose tissue weight (p < 0:001) and food intake (p < 0:05) were observed only in the latter method. The results of changes in plasma leptin concentration and body weight presented here demonstrated that a supraphysiological dose of leptin by peripheral administration was required to lead to significant reductions in food intake and body weight in normal mice, and the potency of the weight-reducing effects of leptin was directly related to the relative rise in plasma leptin concentration after gene transfer in vivo as suggested by Halaas et al. [18]. The significant reduction in epididymal white fat after gene transfer by hydrodynamicsbased transfection implied that an important role of peripheral leptin might be to increase lipid catabolism through sympathetic neuron stimulation primarily in white, and possibly brown adipose tissue as suggested previously [19]. Thus, our data presented here confirmed that both electroporation-mediated leptin gene transfer into leg muscle and hydrodynamics-based gene transfer were effective approaches for systemic delivery of recombinant leptin to the central nervous system, and that the efficiency of gene transfer in hydrodynamics-based gene delivery was markedly higher than that in electroporation at least within the first week after transfection.
References [1] Y. Zhang, R. Proenca, M. Maffei, L. Leopold, J.M. Friedman, Positional cloning of the mouse obese gene and human homologue, Nature 372 (1994) 425–432. [2] S. Collins, C.M. Kuhn, A.E. Petro, A.G. Swick, B.A. Chrunky, R.S. Surwit, Role of leptin in fat regulation, Nature 380 (1996) 677–679. [3] Y.-T. Zhou, M. Shimabukuro, K. Koyama, Y. Lee, M.-Y. Wang, C.B. Newgard, R.H. Unger, Induction by leptin of uncoupling protein-2 and enzymes of acid oxidation, Proc. Natl. Acad. Sci. USA 94 (1997) 6386–6390. [4] S.G. Hassink, E. Lancey, D.V. Sheslow, S.M. Smith-Kirwin, D.M. O’Conner, R.V. Considine, I. Opentanova, K. Dostal, M.L. Spear, K. Leef, A.R. Spitzer, V.L. Funanage, Placental protein: an important new growth factor in intrauterine and neonatal development, Pediatrics 100 (1997) E1. [5] H. Masuzaki, Y. Ogawa, N. Sagowa, K. Hosoda, T. Matsumoto, H. Mise, Y. Yoshimasa, I. Tanaka, T. Mori, K. Nakao, Nonadipose tissue production of leptin: leptin as a novel placentaderived hormone in humans, Nat. Med. 9 (1997) 1029–1033. [6] M.J.M. Lewin, A. Bado, Gastric leptin, Microsc. Res. Tech. 53 (2001) 372–376. [7] L.A. Campfied, F.J. Smith, Y. Guisez, R. Devos, P. Burn, Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks, Science 269 (1995) 546–549. [8] J.L. Halaas, K.S. Gajiwala, M. Maffei, S.L. Cohen, B.T. Chait, D. Rabinowitz, R.L. Lallone, S.K. Burley, J.M. Friedmam, Weightreducing effects of the plasma protein encoded by the obese gene, Science 269 (1995) 543–546. [9] M.A. Pelleymounter, M.J. Cullen, M.B. Baker, R. Hetht, D. Winters, T. Boone, F. Collins, Effects of the obese gene product on body weight regulation in ob/ob mice, Science 269 (1995) 540– 543. [10] D.S. Weigle, T.R. Bukowski, D.C. Foster, S. Holderman, J.M. Kramer, G. Lasser, C.E. Lofton-Day, C. Raymond, J.L. Kuijper, Recombinant OB protein reduces feeding and body weight in the ob/ob mice, J. Clin. Invest. 96 (1995) 2065–2070. [11] K. Koyama, G. Chen, M.Y. Wang, Y. Lee, M. Shimabukuro, C.B. Newgard, R.H. Unger, b-Cell function in normal rats made chronically hyperleptinemic by adenovirus–leptin gene therapy, Diabetes 46 (1997) 1276–1279. [12] F.J. Novo, D.C. Gorecki, G. Goldspink, K.D. MacDermot, Gene transfer and expression of human b-galactosidase from mouse muscle in vitro and in vivo, Gene Ther. 4 (1997) 489–492.
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445 [13] A. Marti, F.J. Novo, E. Martinez-Anso, M. Zaratiegui, M. Aguado, A.J. Martunez, Leptin gene transfer into muscle increases lipolysis and oxygen consumption in white fat tissue in ob/ob mice, Biochem. Biophys. Res. Commun. 246 (1998) 859– 862. [14] M.A. Morsy, M.C. Gu, S. Motzel, J. Zhao, J. Lin, Q. Su, H. Allen, L. Franlin, R.J. Parks, F.L. Graham, K.S. Kochane, A.J. Bett, An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene, Appl. Biol. Sci. 95 (1998) 7866–7871. [15] T. Muramatsu, A. Nakamura, H.-M. Park, In vivo electroporation: a. powerful and convenient means of nonviral gene transfer to tissues of living animals, Int. J. Mol. Med. 1 (1998) 55–62.
445
[16] T. Muramatsu, S. Arakawa, K. Fukazawa, Y. Fujiwara, T. Yoshida, R. Sasaki, H.-M. Park, In vivo gene electroporation in skeletal muscle with special reference to duration of gene expression, Int. J. Mol. Med. 7 (2001) 37–42. [17] F. Liu, Y. Song, D. Liu, Hydrodynamic-based transfection in animal by systemic administration of plasmid DNA, Gene Ther. 6 (1999) 867–870. [18] J.L. Halaas, C. Boozer, W.J. Blair, N. Fidahusein, D.A. Denton, J.M. Frieman, Physiological response to long-term peripheral and central leptin infusion in lean and obese mice, Proc. Natl. Acad. Sci. USA 94 (1997) 8878–8883. [19] K. Rahmouni, W.G. Haynes, Leptin signaling pathways in the central nervous system: interactions between neuropeptide Y and melanocortins, BioEssays 23 (2001) 1095–1099.
Effects of leptin gene expression in mice in vivo by electroporation and hydrodynamics-based gene delivery Lan Xiang,a Atsushi Murai,a Kunio Sugahara,b Akihiro Yasui,c and Tatsuo Muramatsua,* a
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan b Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan c Department of Surgery, Aichi-Saiseikai Hospital, Nagoya 451-0052, Japan Received 9 June 2003
Abstract In vivo electroporation and hydrodynamics-based gene delivery were utilized to test the effect of leptin gene transfer on food intake, and body and fat weights of mice. Gene transfer of pVRmob by electroporation caused a significant reduction in body weight compared with the control counterpart (p < 0:05), although a lesser effect was found in food intake, and the weights of interscapular brown and epididymal fat by electroporation. As might be expected, the hydrodynamics-based transfection method significantly reduced body weight over 1 week post-transfection (p < 0:05). Furthermore, epididymal fat was decreased by 50% at 1 week after gene transfer (p < 0:001). These results suggest that both electroporation and hydrodynamics-based gene delivery may be effective approaches for systemic delivery of recombinant leptin to the central nervous system, and that the efficiency of gene transfer in hydrodynamics-based gene delivery was markedly higher than that in electroporation at least within the first week after transfection. Ó 2003 Elsevier Inc. All rights reserved. Keywords: In vivo electroporation; Hydrodynamics-based gene delivery; Leptin expression vector; Food intake; Body weight
Leptin, product of the obese (ob) gene, functions to regulate food intake and energy expenditure as a hormone synthesized in adipose tissue through targeting the receptor in hypothalamus [1–3]. To a certain extent, placental, gastric, and neonatal brown adipocytes can also synthesize and secrete this hormone [4–6]. The major function of leptin produced in these tissues is to control meal size. Administration of recombinant leptin protein per se in ob/ob mice is known to result in reduced body weight and food intake, and increased metabolic rate [7–10]. However, since exogenous leptin administration would diminish these hormonal effects within a short period, daily injection of the hormone is required. Instead of administrating hormone per se, in vivo gene transfer not only mimicked but also prolonged those exogenous hormonal effects [11–16]. Moreover, such in vivo gene transfer methodology is expected to *
Corresponding author. Fax: +81-52-789-4077. E-mail address: [email protected] (T. Muramatsu). 0006-291X/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01172-0
bring about similar effects with remarkably low cost. Among in vivo gene transfer means, virus vector methods would impose an increased possibility of biohazard and result in diminished effects after repeated use due probably to immunogenicity evoked in host animals and humans. In this respect, physical in vivo methods are considered safer and have an advantage over the virus vector ones, although no report on leptin gene transfer has been published so far. In the present study, in vivo electroporation and hydrodynamics-based gene delivery were utilized to transfer leptin genes, and the effects on food intake, growth, and energy metabolism in mice were investigated.
Materials and methods Plasmid construction. The expression vector, pVRmob, was constructed by substituting the mouse leptin gene excised from pBluescript-mob (kindly provided by Dr. J.M. Friedman, Rockefeller
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445 University, USA) for the mouse erythropoietin gene in pVRmEPO (kindly provided by Dr. J.M. Leiden, Chicago University, USA) through restriction and ligation reactions. Experimental animals. Male ICR strain mice at 6–8 weeks of age were used as experimental animals. They were maintained in a clean room at 23 1 °C with a 12L/12D photoperiodic cycle, fed a commercial diet (MR stock, Nihon Nosan, Yokohama, Japan) ad libitum, and freely received water during the experimental period. The mice were cared for under Guideline of Animal Experimentation, laid down by the Committee of Experimental Animal Care, Nagoya University. Gene electroporation. The mice were anesthetized by injecting with pentobarbital intraperitoneally at 25 mg/kg body weight. Two incisions approximately 2 cm long were made in the skin around the leg muscle, M. gastrocnemius, of left and right legs. The two leg muscles were exposed and 500 lg plasmids (pVR empty vector or pVRmob expression vector) in combination with 20 lg pEGFP-C1 (Clontech, California, CA) were injected with a syringe. Square electric pulses were applied six times with an electroporator (CUY21, NEPA Gene, Ichikawa, Japan) by using a pair of pinecette-type electrodes at 25 V with the loading period of 100 ms/pulse. Then, the skin was stitched with no. 3 silk suture. After electroporation, the mice were maintained for up to the subsequent 4 weeks. Food intake and body weight were recorded daily for the entire experimental period. At designated time points during the experiment, blood was taken in a heparinized hematocrit tube for analysis of plasma leptin concentration. At the end of the experiment, treated mice were killed by neck dislocation and leg muscle was removed for analyses of GFP expression, leptin mRNA by RT-PCR, and leptin by Western blotting. GFP expression in the leg muscle was observed with a fluorescent microscope (SZX-RFL2, Olympus, Tokyo, Japan). Epididymal white fat and interscapular brown fat were removed quickly and weighed. Hydrodynamics-based gene delivery. Injection of the plasmid was performed as described by Liu et al. [17]. Briefly, the male mice were anesthetized by injecting with pentobarbital intraperitoneally at 25 mg/ kg body weight. Then, 12.5 lg DNA (pVR empty vector or pVRmob expression vector) in lactated Ringer’s solution (0.1 ml/g body weight) was injected into the tail vein using a syringe with a 27-gauge needle within 10 s. After hydrodynamics-based gene delivery, the mice were maintained for up to the subsequent 2 weeks. Food intake and body weight were recorded daily for the entire experimental period. At designated time points during the experiment, blood was taken as in the electroporation experiment for analysis of plasma leptin concentration. At the end of the experiment, treated mice were killed by neck dislocation and liver was removed for analysis of GFP expression, which was determined with a fluorescent microscope (SZX-RFL2, Olympus, Tokyo, Japan). Epididymal white fat and interscapular brown fat were removed quickly and weighed as in the electroporation experiment. RT-PCR assay. Approximately 50 mg electroporation-treated muscle samples of the transfected area were taken at 3 days after gene transfer. Total RNA was isolated using the Trizol reagent procedure (Life Technologies, Rockville, MD) and RNA content was quantitated spectrophotometrically at 260 nm. RT-PCR was done with a commercially available kit (Takara RNA PCR kit (AMV), Otsu, Japan) to reverse transcribe 1 lg total RNA isolated from the muscle sample using an oligo(dT) adaptor primer, with Perkin–Elmer DNA Thermal Cycler (Takara, Otsu, Japan) and Taq polymerase (Promega, Madison, WI). The mouse leptin-specific primers used for the PCR were as follows: sense, 50 -GAC CCC TGT GTC GGT TCC TGT-30 ; antisense 50 -GCT TCT GCA GGC CAC TGG TCT-30 . Western blot analysis. Approximately 0.2 g electroporation-treated muscle samples were taken at 3 days after gene transfer and placed in a 5 ml centrifuge tube to which 2 ml of lysis buffer (500 mM Tris–HCl, 10% SDS, 50% glycerol, and 500 mM DTT, pH adjusted to 6.8) was added. After homogenization and sonication, they were centrifuged at 12,000g at 4 °C for 3 min and the supernatant was transferred to a new tube. Subsequently, protein concentration was measured by Lowry
441
method with a spectrophotometer at 550 nm. The sample protein at 100 lg was run on a 15% SDS–PAGE gel. The proteins were transferred to a PVDF membrane and immunoblotted with a leptin antibody (Sigma, St. Louis, MO, USA), followed by incubation with the second antibody, anti-rabbit IgG conjugated with horseradish peroxidase (Promega, Madison, WI). Chemiluminescence was measured with a Light-capture (AE-6955, ATTO, Tokyo, Japan). Enzyme-linked immunosorbent assay. The plasma samples at 2 and 4 weeks after gene transfer by electroporation, and at 2, 4, 6, 10, and 14 days after gene transfer by hydrodynamics-based gene delivery were obtained by centrifuging the blood at 6000g at room temperature for 5 min and stored at )20 °C until analysis. The plasma samples were assayed for leptin concentration using a commercially available ELISA kit (mouse leptin ELISA kit, Morinaga, Yokohama, Japan) according to the manufacturer’s protocol. Statistical analysis. Statistical treatment on the data was done by a one-way analysis of variance using a commercially available statistical package, SAS (SAS Institute, Cary, NC), and the significance of difference between means was examined by a protected least significant difference method. Results were expressed as means SEM. A value of P < 0:05 was considered significant.
Results GFP expression in animals after electroporation and hydrodynamics-based gene delivery GFP expression in leg muscle and liver after leptin gene transfer by electroporation and hydrodynamicsbased gene delivery, respectively, is presented in Fig. 1. Clear GFP-specific fluorescence signals were detected along the muscle fibers by electroporation and hepatocytes by hydrodynamics-based gene delivery. This implied that the simultaneously transferred pVRmob was successfully expressed in both the muscle and liver. Detection of leptin mRNA and leptin in leg muscle The results of RT-PCR and Western blot from the electroporation-treated muscle are shown in Figs. 2A and B, respectively. The specific bands for the leptin mRNA and leptin protein of 372 bp and 16 kDa size, respectively, were observed. These results suggested that leptin mRNA and leptin protein were produced adequately in leg muscle after gene transfer in vivo by electroporation. Effects of in vivo electroporation on plasma leptin concentration, food intake, and body weight Fig. 3 displays the values for plasma leptin concentration after gene transfer by electroporation. At both 2 and 4 weeks, the leptin gene transfer group had higher plasma leptin concentrations than did the control counterpart and significant difference was detected at 2 weeks (p < 0:05). There was a significant increase both in the control and pVRmob groups from 2 to 4 weeks after gene transfer (p < 0:05). This age-dependent
442
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445
Fig. 1. GFP gene expression in the mouse leg muscle after in vivo gene transfer by electroporation (A) and in the liver by hydrodynamics-based gene delivery (B). The GFP expression plasmid, pEGFP-C1, was co-transferred at a dose of 20 lg per animal in combination with pVRmob. GFP-specific fluorescence signals were detected with a fluorescent microscope at 7 days after in vivo gene electroporation in muscle (A) and 1 day after hydrodynamics-based gene delivery (B). The arrows show fluorescent-positive myotubes (A) or liver cells (B). Scale is given by a horizontal bar.
Fig. 2. Analysis of pVRmob transcript and the resultant protein products. (A) RT-PCR amplified leptin mRNA in the mouse leg muscle to which pVRmob was transferred in vivo by electroporation at a dose of 500 lg per animal. Lanes: M, 100 bp marker; 1 and 2, control muscle; 3–5, pVRmob transferred muscle. (B) Western blotting of leptin protein in the mouse leg muscle after gene transfer. Lanes: 1, 2, control muscle; 3–5, pVRmob transferred muscle.
observed at 4, 5, and 7 days after in vivo gene transfer in the pVRmob group compared with the control counterpart (p < 0:05). Daily food intake was not significantly different between the two treatment groups. The weights of epididymal, white fat, interscapular brown fat, and liver were little affected by the leptin gene transfer (p > 0:05, data not shown). Effects of hydrodynamics-based gene delivery on plasma leptin concentration, food intake, body weight, and adipose tissue weight
Fig. 3. Changes in plasma leptin concentration after leptin gene transfer in vivo by electroporation at a dose of 500 lg per animal. Vertical bars represent SEM of five mice. *Significantly different at p < 0:05; ns, not significantly different (p > 0:05).
increase may probably be brought about by gross increases in adipose weight due to aging, leading to increased production of leptin in the plasma. Fig. 4 gives the values for body weight and food intake after gene transfer by electroporation. Body weight tended to be decreased and significant difference was
The leptin concentration in plasma after in vivo gene transfer by hydrodynamics-based gene delivery is given in Fig. 5. At 2 days post-transfection, a remarkable increase was detected by a factor of 40 in the leptin gene transferred group compared with the control group, although leptin concentration was sharply decreased afterwards. Significant differences in plasma leptin were detected in the pVRmob group compared with the control counterpart at 2 days (p < 0:001), and 4 and 10 days (p < 0:05). Fig. 6 presents the values for food intake and body weight after gene transfer by hydrodynamics-based gene delivery. Body weight was significantly reduced at 2 days
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445
443
Fig. 4. Changes in body weight and food intake of mice after transferring pVRmob gene in vivo by electroporation at a dose of 500 lg per animal. Each point represents means SEM of five mice. *Significantly different from the control at the same time point at p < 0:05 (*).
and the lowered body weight was maintained for the first 7 days (p < 0:05). The significant reduction in food intake was detected only at 2 days after gene transfer (p < 0:05) and the food intake was quickly restored to the normal level. Fig. 7 evaluates the values for epididymal white fat and interscapular brown fat. These weights tended to be reduced at 1 week and the reduction in epididymal white fat weight reached statistical significance in the treated group compared with the control counterpart (p < 0:001).
Discussion
Fig. 5. Changes in plasma leptin concentration after leptin gene transfer in vivo by hydrodynamics-based gene delivery at a dose of 12.5 lg per animal. Each point represents means SEM of three mice. *, ** Significantly different from the control at the same time point at p < 0:05 (*) and p < 0:001 (**).
In the present study, to monitor whether a foreign gene could be transferred in vivo by electroporation and hydrodynamics-based gene delivery, a GFP expression plasmid, pEGFP-C1, was transferred. The results of strong GFP expression indicated that in vivo gene transfer was successfully completed. RT-PCR
Fig. 6. Changes in body weight and food intake of mice after transferring pVRmob gene in vivo by hydrodynamics-based gene delivery at a dose of 12.5 lg per animal. Each point represents means SEM of five mice. * Significantly different from the control at the same time point at p < 0:05 (*); ns, not significantly different (p > 0:05).
444
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445
In conclusion, the present study provided the first direct evidence of successful leptin gene transfer in mice in vivo by electroporation and hydrodynamics-based gene delivery. These nonviral gene transfer methods would facilitate the better understanding of regulatory mechanisms of leptin on food intake and energy metabolism in vivo.
Acknowledgments We gratefully acknowledge Dr. J.M. Friedman, Rockefeller University, and Dr. J.M. Leiden, Chicago University, USA, for gifts of pBluescript-mob and pVRmEPO plasmids, respectively. This work was in part supported by a Grant-in-Aid for Scientific Research (B) (2) (No. 15380192) from the Japan Society for the Promotion of Science. Fig. 7. Weights of epdidymal white fat and interscapular brown fat of mice to which pVRmob was transferred in vivo by hydrodynamicsbased gene delivery at a dose of 12.5 lg per animal. The adipose tissue weights were measured at 1 week after gene transfer. Vertical bars represent SEM of five mice. ** Significantly different at p < 0:001; ns, not significantly different (p > 0:05).
and Western blotting analyses demonstrated that the simultaneously transfected leptin gene was adequately transcribed and translated. Effects of leptin gene transfer on reduced food intake and decreased body weight were also observed, which were in good agreement with those of leptin administration in the previous studies [9–13]. However, the extent of reduction in body weight was smaller by electroporation (3.5%) than by hydrodynamics-based transfection (6.3%). Significant reductions in adipose tissue weight (p < 0:001) and food intake (p < 0:05) were observed only in the latter method. The results of changes in plasma leptin concentration and body weight presented here demonstrated that a supraphysiological dose of leptin by peripheral administration was required to lead to significant reductions in food intake and body weight in normal mice, and the potency of the weight-reducing effects of leptin was directly related to the relative rise in plasma leptin concentration after gene transfer in vivo as suggested by Halaas et al. [18]. The significant reduction in epididymal white fat after gene transfer by hydrodynamicsbased transfection implied that an important role of peripheral leptin might be to increase lipid catabolism through sympathetic neuron stimulation primarily in white, and possibly brown adipose tissue as suggested previously [19]. Thus, our data presented here confirmed that both electroporation-mediated leptin gene transfer into leg muscle and hydrodynamics-based gene transfer were effective approaches for systemic delivery of recombinant leptin to the central nervous system, and that the efficiency of gene transfer in hydrodynamics-based gene delivery was markedly higher than that in electroporation at least within the first week after transfection.
References [1] Y. Zhang, R. Proenca, M. Maffei, L. Leopold, J.M. Friedman, Positional cloning of the mouse obese gene and human homologue, Nature 372 (1994) 425–432. [2] S. Collins, C.M. Kuhn, A.E. Petro, A.G. Swick, B.A. Chrunky, R.S. Surwit, Role of leptin in fat regulation, Nature 380 (1996) 677–679. [3] Y.-T. Zhou, M. Shimabukuro, K. Koyama, Y. Lee, M.-Y. Wang, C.B. Newgard, R.H. Unger, Induction by leptin of uncoupling protein-2 and enzymes of acid oxidation, Proc. Natl. Acad. Sci. USA 94 (1997) 6386–6390. [4] S.G. Hassink, E. Lancey, D.V. Sheslow, S.M. Smith-Kirwin, D.M. O’Conner, R.V. Considine, I. Opentanova, K. Dostal, M.L. Spear, K. Leef, A.R. Spitzer, V.L. Funanage, Placental protein: an important new growth factor in intrauterine and neonatal development, Pediatrics 100 (1997) E1. [5] H. Masuzaki, Y. Ogawa, N. Sagowa, K. Hosoda, T. Matsumoto, H. Mise, Y. Yoshimasa, I. Tanaka, T. Mori, K. Nakao, Nonadipose tissue production of leptin: leptin as a novel placentaderived hormone in humans, Nat. Med. 9 (1997) 1029–1033. [6] M.J.M. Lewin, A. Bado, Gastric leptin, Microsc. Res. Tech. 53 (2001) 372–376. [7] L.A. Campfied, F.J. Smith, Y. Guisez, R. Devos, P. Burn, Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks, Science 269 (1995) 546–549. [8] J.L. Halaas, K.S. Gajiwala, M. Maffei, S.L. Cohen, B.T. Chait, D. Rabinowitz, R.L. Lallone, S.K. Burley, J.M. Friedmam, Weightreducing effects of the plasma protein encoded by the obese gene, Science 269 (1995) 543–546. [9] M.A. Pelleymounter, M.J. Cullen, M.B. Baker, R. Hetht, D. Winters, T. Boone, F. Collins, Effects of the obese gene product on body weight regulation in ob/ob mice, Science 269 (1995) 540– 543. [10] D.S. Weigle, T.R. Bukowski, D.C. Foster, S. Holderman, J.M. Kramer, G. Lasser, C.E. Lofton-Day, C. Raymond, J.L. Kuijper, Recombinant OB protein reduces feeding and body weight in the ob/ob mice, J. Clin. Invest. 96 (1995) 2065–2070. [11] K. Koyama, G. Chen, M.Y. Wang, Y. Lee, M. Shimabukuro, C.B. Newgard, R.H. Unger, b-Cell function in normal rats made chronically hyperleptinemic by adenovirus–leptin gene therapy, Diabetes 46 (1997) 1276–1279. [12] F.J. Novo, D.C. Gorecki, G. Goldspink, K.D. MacDermot, Gene transfer and expression of human b-galactosidase from mouse muscle in vitro and in vivo, Gene Ther. 4 (1997) 489–492.
L. Xiang et al. / Biochemical and Biophysical Research Communications 307 (2003) 440–445 [13] A. Marti, F.J. Novo, E. Martinez-Anso, M. Zaratiegui, M. Aguado, A.J. Martunez, Leptin gene transfer into muscle increases lipolysis and oxygen consumption in white fat tissue in ob/ob mice, Biochem. Biophys. Res. Commun. 246 (1998) 859– 862. [14] M.A. Morsy, M.C. Gu, S. Motzel, J. Zhao, J. Lin, Q. Su, H. Allen, L. Franlin, R.J. Parks, F.L. Graham, K.S. Kochane, A.J. Bett, An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene, Appl. Biol. Sci. 95 (1998) 7866–7871. [15] T. Muramatsu, A. Nakamura, H.-M. Park, In vivo electroporation: a. powerful and convenient means of nonviral gene transfer to tissues of living animals, Int. J. Mol. Med. 1 (1998) 55–62.
445
[16] T. Muramatsu, S. Arakawa, K. Fukazawa, Y. Fujiwara, T. Yoshida, R. Sasaki, H.-M. Park, In vivo gene electroporation in skeletal muscle with special reference to duration of gene expression, Int. J. Mol. Med. 7 (2001) 37–42. [17] F. Liu, Y. Song, D. Liu, Hydrodynamic-based transfection in animal by systemic administration of plasmid DNA, Gene Ther. 6 (1999) 867–870. [18] J.L. Halaas, C. Boozer, W.J. Blair, N. Fidahusein, D.A. Denton, J.M. Frieman, Physiological response to long-term peripheral and central leptin infusion in lean and obese mice, Proc. Natl. Acad. Sci. USA 94 (1997) 8878–8883. [19] K. Rahmouni, W.G. Haynes, Leptin signaling pathways in the central nervous system: interactions between neuropeptide Y and melanocortins, BioEssays 23 (2001) 1095–1099.