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Hyperleptinemia in Female Patients with Ossification of Spinal Ligaments
Biochemical and Biophysical Research Communications 267, 752–755 (2000) doi:10.1006/bbrc.1999.2027, available online at http://www.idealibrary.com on
Hyperleptinemia in Female Patients with Ossification of Spinal Ligaments Yoshiharu Shirakura, 1 Toshihiro Sugiyama, Hiroshi Tanaka, Toshihiko Taguchi, and Shinya Kawai Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan
Received December 7, 1999
In order to examine the involvement of leptin in the ossification of spinal ligaments (OSL), the present study examined (i) serum levels of leptin and insulin in OSL patients and controls, (ii) serum leptin levels in children of OSL females with severe obesity, (iii) the expression of leptin receptor mRNA in human spinal ligaments, and (iv) effects of leptin on cultured human ligament cells. In the OSL females, serum leptin levels were significantly higher than those of the control females, and the levels were positively correlated to the serum insulin levels, while in the control females, there was a tendency of inverse correlation. The daughters of OSL females with severe obesity also had high serum leptin levels, although they had not developed OSL. The expression of leptin receptor mRNA was confirmed in the ligaments, but leptin did not influence the alkaline phosphatase activity nor procollagen type I carboxyl-terminal peptide content of the ligament cells. These findings suggest that leptin is involved genetically and indirectly with the pathogenesis of OSL in female patients. © 2000 Academic Press
Ossification of spinal ligaments (OSL) is characterized by a heterotopic bone formation in the spinal ligament that is normally composed of fibrous tissues, and OSL is one of the causes of compression myelopathy. The pathogenesis of OSL is still unknown, though the involvement of genetic factors [1] and disorders in glucose metabolism [2, 3] has been reported. Zucker fatty (fa/fa) rats [4] are hereditary obesity rats, develop OSL, and have an aberration of the leptin receptor gene [5– 8]. Leptin is encoded in the obese (ob) gene [9], secreted by adipose tissues, and involved with the maintenance of body weight. The leptin-knockout ob/ob mice develop increases of body weight and insulin level in the blood, and these are improved when exogTo whom correspondence should be addressed. Fax: ⫹81-836-222267. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
enous leptin is administered [10 –12]. In a human family with leptin receptor abnormalities [13], the family members developed obesity and their leptin levels in the blood were high. The present study examined our hypothesis that leptin could be one of the causative factors of human OSL. MATERIALS AND METHODS Measurements of serum leptin and insulin levels. Informed consent of each subject and institutional permission were obtained before starting this study. Subjects were 49 OSL patients and 57 controls. The controls did not have OSL in roentgenography or any abnormalities in bone metabolism. Serum leptin levels were also examined in children of 2 female patients who developed OSL at an early age and who had severe obesity. Venous blood was collected at 2 h after breakfast, and the serum was stored at ⫺80°C until use. Serum leptin level was measured with radioimmunoassay (Human Leptin RIA Kit, Linco Research Inc., St. Charles, MO) [14]. Because gender and the volume of adipose tissue could influence leptin, the measured serum leptin levels were corrected by body mass index (BMI), and then compared in each gender group [15, 16]. Minimum detection limit of serum leptin level was 0.5 ng/ml, and the coefficient of variation was 4.5%. Serum insulin level was measured with microparticle enzyme immunoassay (AxSYM insulin assay kit, Dainabot Co., Ltd., Tokyo). Minimum detection limit of serum insulin level was 0.8 U/ml, and the coefficient of variation was 5.5%. Expression analysis of leptin receptor mRNA. Human yellow ligaments (3 females without OSL) resected at laminoplasty were used [17]. It is difficult to use yellow ligaments of OSL patients because they have been already ossified at the operation. As a positive control, human adipose tissue was used. Total RNA was extracted by the guanidinium thiocyanate-phenol-chloroform extraction method (TRIzol Reagent, Gibco BRL, NY) [18]. First strand cDNA was synthesized from 5 g of total RNA using oligo-dT as a primer and AMV reverse transcriptase (cDNA Synthesis Kit, Boehringer Mannheim GmbH, Mannheim). For the amplification reaction, the following set of primers was synthesized according to the published sequence of the human leptin receptor [19], i.e., sense: 5⬘-CATTTTATCCCCATTGAGAAGTA-3⬘, and antisense: 5⬘-CTGAAAATTAAGTCCTTGTGCCCAG-3⬘. In PCR analysis, 15 l of the mixture of the following materials was used: the above-mentioned reverse transcription reaction mixture, 1.5 mM dNTPs, 0.375 Units of Taq DNA polymerase (Boehringer Mannheim GmbH, Mannheim), and 3.75 pmol of each primer. After pre-denaturation at 94°C for 5 min, amplification was carried out with the following thermocycling conditions: one cycle
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Characteristics of the Subjects for Measurements of Serum Leptin and Insulin Levels Female
Male
OSL patients Controls OSL patients Controls (n ⫽ 17) (n ⫽ 20) (n ⫽ 32) (n ⫽ 37) Age (years) 56 ⫾ 3 58 ⫾ 4 61 ⫾ 2 61 ⫾ 2 Height (m) 1.52 ⫾ 0.02 1.53 ⫾ 0.02 1.65 ⫾ 0.01 1.63 ⫾ 0.01 Weight (kg) 59.4 ⫾ 3.3 54.9 ⫾ 1.5 63.8 ⫾ 1.8 63.1 ⫾ 1.3 BMI (kg 䡠 m ⫺2) 25.4 ⫾ 1.1 23.4 ⫾ 0.4 23.3 ⫾ 0.6 23.7 ⫾ 0.5 Note. OSL, ossification of spinal ligaments. BMI, body mass index. Figures represent mean ⫾ SD.
consisted of 30 seconds denaturation at 94°C, 30 seconds annealing at 55°C, and 30 seconds extension at 72°C. After completing 35 cycles, an additional 5-min extension at 72°C was carried out in the GeneAmp PCR System Model 9600 (Perkin-Elmer Corp., Norwalk, CT). Amplified products were electrophoresed using 3% (w/v) agarose gel and stained with ethidium bromide. Effects of leptin on ligament cells. Human yellow ligaments (10 females) resected at surgery were used [17]. The ligaments were cut into pieces in ␣-minimum essential medium (␣-MEM), washed with ␣-MEM, placed in 350 cm 2 flasks containing ␣-MEM supplemented with 20% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 g/ml), and then incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2. The explants were cultured for 14 days with the medium change at the 7th day, then outgrowth cells were detached by 0.05% trypsin and 0.05% EDTA for 5 min at 37°C. The collected cells were seeded in 12-well plates containing ␣-MEM supplemented with 10% heat inactivated FBS, penicillin (100 U/ml), and streptomycin (100 g/ml), at a density of FIG. 2. Relationship between serum leptin level corrected by body mass index (BMI) and serum insulin level. (A) In the ossification of spinal ligaments (OSL) females, there was a positive correlation. (B) In the control females, there was a tendency of inverse but no significant correlation.
5 ⫻ 10 3 cells/cm 2. ␣-MEM, FBS, penicillin, streptomycin, trypsin and EDTA were all purchased from Gibco BRL (NY). When the cells reached confluent level, the medium was renewed, then the cells were stimulated with recombinant human leptin (R&D Systems, Minneapolis, MN) at 0, 1, 10, or 100 ng/ml, and incubated for 3, 6, or 9 days. The medium was changed once every 3 days. Alkaline phosphatase (AP) activities of the cell layer were assessed, and the data were standardized by referring to the protein content, as described previously [20, 21]. Procollagen type I carboxyl-terminal peptide (PICP) content released into the medium for 24 h was assessed using a radioimmunoassay kit (Chugai Pharmaceutical Co., Ltd., Tokyo) after the medium had been replaced with the serum-free medium (ASF-301, Ajinomoto Co., Tokyo), and the data were also standardized by referring to the protein content.
FIG. 1. Serum leptin levels corrected by body mass index (BMI) in the ossification of spinal ligaments (OSL) patients and controls. Only in the OSL females, the leptin levels were significantly higher than the control females.
Statistical analysis. To compare OSL patients and controls, Student’s t-test was done for age, height, body weight, BMI, and serum leptin level corrected by BMI. The relationship between the serum leptin level and the serum insulin level was analyzed using Pearson’s correlation analysis. Student’s t-test was also done for AP activity and PICP content between the cells with or without recombinant human leptin stimulation at various concentrations. All these analyses were performed with a significance of P ⬍ 0.05.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2
Serum Leptin Level and Body Mass Index (BMI) of Two Female Patients Who Developed OSL at an Early Age and Who Had Severe Obesity, and Their Children
1 1’s daughter 2 2’s daughter Control females
Age (years)
BMI (kg 䡠 m ⫺2)
Leptin/BMI (ng 䡠 ml ⫺1/kg 䡠 m ⫺2)
40 17 46 25 58 ⫾ 4*
31.6 21.0 40.6 37.0 23.4 ⫾ 0.4*
0.769 0.729 0.611 1.386 0.220 ⫾ 0.018*
* Mean ⫾ SD.
RESULTS Characteristics of the subjects for measurements of serum leptin and insulin levels are shown in Table 1. There was no significant difference in the two groups. Serum leptin level corrected by BMI in the OSL females (mean ⫾ SD, 0.426 ⫾ 0.039 ng 䡠 ml ⫺1/kg 䡠 m ⫺2) was significantly (P ⬍ 0.01) higher than the control females (0.220 ⫾ 0.018 ng 䡠 ml ⫺1/kg 䡠 m ⫺2) (Fig. 1). On the other hand, in male subjects, serum leptin levels corrected by BMI were 0.144 ⫾ 0.013 ng 䡠 ml ⫺1/kg 䡠 m ⫺2 and 0.144 ⫾ 0.012 ng 䡠 ml ⫺1/kg 䡠 m ⫺2 for the OSL patients and controls, respectively. The levels were not significantly different between two male groups. In the OSL females, serum leptin level was positively correlated to the serum insulin level (r ⫽ 0.509, P ⬍ 0.05), while in the control females, there was a tendency of inverse but no significant (r ⫽ ⫺0.390, P ⫽ 0.09) correlation between serum levels of leptin and insulin (Fig. 2). The daughters of two female OSL patients had high serum leptin levels, though they did not have OSL (Table 2). RT-PCR analysis demonstrated the expression of leptin receptor mRNA in female spinal ligaments (Fig. 3), however AP activity and PICP content were not
FIG. 4. Effect of recombinant human leptin (rh-leptin) on the cultured female ligament cells. Alkaline phosphatase (AP) activity and procollagen type I carboxyl-terminal peptide (PICP) content of the cells stimulated with rh-leptin at 1, 10, or 100 ng/ml were compared to those with rh-leptin at 0 ng/ml. AP activity and PICP content in the cultured human ligament cells were not affected by exogenous rh-leptin.
affected by exogenous recombinant human leptin in the cultured female spinal ligament cells (Fig. 4). DISCUSSION
FIG. 3. RT-PCR findings. A single band of 271 bp (arrow) was obtained in the surgically resected female spinal ligaments. The lanes represent a 39-year-old female (A), a 61-year-old female (B), and a 66-year-old female (C). The result in human adipose tissue is also shown in lane (D) as a positive control.
The present study demonstrated that OSL females had a significantly high serum leptin level, and their leptin levels positively correlated to the serum insulin levels. In the control females, there was a tendency of inverse correlation between the serum levels of leptin and insulin, though it was not significant. It has been reported that alterations in plasma leptin concentrations might play a role in the etiology of insulin resistance [22] or cardiovascular disease [23]. Our findings suggest the involvement of leptin with the occurrence of female OSL.
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The involvement of genetic factors has been suggested in OSL [1]. The daughters of 2 female patients who developed OSL at an early age and who had severe obesity as observed in the Zucker fatty (fa/fa) rats also had a high serum leptin level, though they had not developed OSL. These findings suggest that leptin is genetically involved with the pathogenesis of female OSL. Genetic disorders in the leptin system may be present only in females, and the mechanism of OSL development could be different according to gender. In the present study, the expression of leptin receptor mRNA was confirmed in the female spinal ligaments, but recombinant human leptin did not affect AP activity nor PICP content of the female ligament cells, which are markers for the bone formation [24, 25]. Another study also reported that leptin didn’t have any significant direct effect in controlling bone cell activity [26]. Leptin was reported to suppress insulin secretion [27], and insulin has been thought to accelerate bone formation [28 –30]. A previous study [2] showed that OSL patients have high serum insulin levels. Therefore, leptin may affect indirectly the occurrence and establishment of female OSL when leptin is not able to suppress insulin because of a certain genetic abnormality. On the other hand, leptin was reported to act on human marrow stromal cells to enhance differentiation to osteoblasts [31], and leptin may also have direct effects on heterotopic bone formation in the human spinal ligaments. The effect of leptin to bone formation is still controversial. ACKNOWLEDGMENT This work was supported by a grant-in-aid from the Investigation Committee on the Ossification of Spinal Ligament, Ministry of Health and Welfare of Japan.
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8. Takaya, K., Ogawa, Y., Isse, N., Okazaki, T., Satoh, N., Masuzaki, H., Mori, K., Tamura, N., Hosoda, K., and Nakao, K. (1996) Biochem. Biophys. Res. Commun. 225, 75– 83. 9. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J. M. (1994) Nature 372, 425– 432. 10. Pelleymounter, M. A., Cullen, M. J., Baker, M. B., Hecht, R., Winters, D., Boone, T., and Collins, F. (1995) Science 269, 540 – 543. 11. Halaas, J. L., Gajiwala, K. S., Maffei, M., Cohen, S. L., Chait, B. T., Rabinowitz, D., Lallone, R. L., Burley, S. K., and Freidman, J. M. (1995) Science 269, 543–546. 12. Campfield, L. A., Smith, F. J., Guisez, Y., Devos, R., and Burn, P. (1995) Science 269, 546 –549. 13. Clement, K., Vaisse, C., Lahlou, N., Cabrol, S., Pelloux, V., Cassuto, D., Gourmelen, M., Dina, C., Chambaz, J., Lacorte, J. M., Basdevant, A., Bougneres, P., Lebouc, Y., Froguel, P., and Guy-Grand, B. (1998) Nature 392, 398 – 401. 14. Ma, Z., Gingerich, R. L., Santiago, J. V., Klein, S., Smith, C. H., and Landt, M. (1996) Clin. Chem. 42, 942–946. 15. Hannan, W. J., Wrate, R. M., Cowen, S. T., and Freeman, C. P. L. (1995) Int. J. Eating Disorders 18, 91–97. 16. Rosenbaum, M., Nicolson, M., Hirsch, J., Heymsfield, S. B., Gallagher, D., Chu, F., and Leibel, R. L. (1996) J. Clin. Endocrinol. Metab. 81, 3424 –3427. 17. Kawai, S., Hattori, S., Oda, H., Yamaguchi, Y., and Yoshida, Y. (1981) Spine 6, 381–387. 18. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156 –159. 19. Taltaglia, L. A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R., Richards, G. J., Campfield, L. A., Clark, F. T., Deeds, J., Muir, C., Sanker, S., Moriatry, A., Moore, K. J., Smutko, J. S., Mays, G. G., Woolf, E. A., Monroe, C. A., and Tepper, R. I. (1995) Cell 83, 1263–1271. 20. Ishida, Y., and Kawai, S. (1993) Bone 14, 85–91. 21. Ishida, Y., and Kawai, S. (1993) J. Bone Miner. Res. 8, 1291– 1300. 22. Zimmet, P., Hodge, A., Nicolson, M., Staten, M., Courten, M., Moore, J., Morawiecki, A., Lubina, J., Collier, G., Alberti, G., and Dowse, G. (1996) BMJ 313 965–969. 23. Leyva, F., Godsland, I. F., Ghatei, M., Proudler, A. J., Aldis, S., Walton, C., Bloom, S., and Stevenson, J. C. (1998) Arterioscler. Thromb. Vasc. Biol. 18, 928 –933. 24. Wlodarski K. H. (1990) Clin. Orthop. 252, 276 –293 25. Parfitt, A. M., Simon, L. S., Villanueva, A. R., and Krane, S. M. (1987) J. Bone Miner. Res. 2, 427– 436 26. Goulding, A., and Taylor, R. W. (1998) Calcif. Tissue Int. 63, 456 – 458 27. Pallett, A. L., Morton, N. M., Cawthorne, M. A., and Emilsson, V. (1997) Biochem. Biophys. Res. Commun. 238, 267–270. 28. Canalis, E. M., Dietrich, J. W., Maina, D. M., and Raisz, L. G. (1977) Endocrinology 100, 668 – 674. 29. Kream, B. E., Smith, M. D., Canalis, E., and Raisz, L. G. (1985) Endocrinology 116, 296 –302. 30. Peck, W. A., and Messinger, K. (1970) J. Biol. Chem. 245, 2722– 2729. 31. Thomas, T., Gori, F., Khosla, S., Jensen, M. D., Burguera, B., and Riggs, B. L. (1999) Endocrinology 140, 1630 –1638.
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Hyperleptinemia in Female Patients with Ossification of Spinal Ligaments Yoshiharu Shirakura, 1 Toshihiro Sugiyama, Hiroshi Tanaka, Toshihiko Taguchi, and Shinya Kawai Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan
Received December 7, 1999
In order to examine the involvement of leptin in the ossification of spinal ligaments (OSL), the present study examined (i) serum levels of leptin and insulin in OSL patients and controls, (ii) serum leptin levels in children of OSL females with severe obesity, (iii) the expression of leptin receptor mRNA in human spinal ligaments, and (iv) effects of leptin on cultured human ligament cells. In the OSL females, serum leptin levels were significantly higher than those of the control females, and the levels were positively correlated to the serum insulin levels, while in the control females, there was a tendency of inverse correlation. The daughters of OSL females with severe obesity also had high serum leptin levels, although they had not developed OSL. The expression of leptin receptor mRNA was confirmed in the ligaments, but leptin did not influence the alkaline phosphatase activity nor procollagen type I carboxyl-terminal peptide content of the ligament cells. These findings suggest that leptin is involved genetically and indirectly with the pathogenesis of OSL in female patients. © 2000 Academic Press
Ossification of spinal ligaments (OSL) is characterized by a heterotopic bone formation in the spinal ligament that is normally composed of fibrous tissues, and OSL is one of the causes of compression myelopathy. The pathogenesis of OSL is still unknown, though the involvement of genetic factors [1] and disorders in glucose metabolism [2, 3] has been reported. Zucker fatty (fa/fa) rats [4] are hereditary obesity rats, develop OSL, and have an aberration of the leptin receptor gene [5– 8]. Leptin is encoded in the obese (ob) gene [9], secreted by adipose tissues, and involved with the maintenance of body weight. The leptin-knockout ob/ob mice develop increases of body weight and insulin level in the blood, and these are improved when exogTo whom correspondence should be addressed. Fax: ⫹81-836-222267. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
enous leptin is administered [10 –12]. In a human family with leptin receptor abnormalities [13], the family members developed obesity and their leptin levels in the blood were high. The present study examined our hypothesis that leptin could be one of the causative factors of human OSL. MATERIALS AND METHODS Measurements of serum leptin and insulin levels. Informed consent of each subject and institutional permission were obtained before starting this study. Subjects were 49 OSL patients and 57 controls. The controls did not have OSL in roentgenography or any abnormalities in bone metabolism. Serum leptin levels were also examined in children of 2 female patients who developed OSL at an early age and who had severe obesity. Venous blood was collected at 2 h after breakfast, and the serum was stored at ⫺80°C until use. Serum leptin level was measured with radioimmunoassay (Human Leptin RIA Kit, Linco Research Inc., St. Charles, MO) [14]. Because gender and the volume of adipose tissue could influence leptin, the measured serum leptin levels were corrected by body mass index (BMI), and then compared in each gender group [15, 16]. Minimum detection limit of serum leptin level was 0.5 ng/ml, and the coefficient of variation was 4.5%. Serum insulin level was measured with microparticle enzyme immunoassay (AxSYM insulin assay kit, Dainabot Co., Ltd., Tokyo). Minimum detection limit of serum insulin level was 0.8 U/ml, and the coefficient of variation was 5.5%. Expression analysis of leptin receptor mRNA. Human yellow ligaments (3 females without OSL) resected at laminoplasty were used [17]. It is difficult to use yellow ligaments of OSL patients because they have been already ossified at the operation. As a positive control, human adipose tissue was used. Total RNA was extracted by the guanidinium thiocyanate-phenol-chloroform extraction method (TRIzol Reagent, Gibco BRL, NY) [18]. First strand cDNA was synthesized from 5 g of total RNA using oligo-dT as a primer and AMV reverse transcriptase (cDNA Synthesis Kit, Boehringer Mannheim GmbH, Mannheim). For the amplification reaction, the following set of primers was synthesized according to the published sequence of the human leptin receptor [19], i.e., sense: 5⬘-CATTTTATCCCCATTGAGAAGTA-3⬘, and antisense: 5⬘-CTGAAAATTAAGTCCTTGTGCCCAG-3⬘. In PCR analysis, 15 l of the mixture of the following materials was used: the above-mentioned reverse transcription reaction mixture, 1.5 mM dNTPs, 0.375 Units of Taq DNA polymerase (Boehringer Mannheim GmbH, Mannheim), and 3.75 pmol of each primer. After pre-denaturation at 94°C for 5 min, amplification was carried out with the following thermocycling conditions: one cycle
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Characteristics of the Subjects for Measurements of Serum Leptin and Insulin Levels Female
Male
OSL patients Controls OSL patients Controls (n ⫽ 17) (n ⫽ 20) (n ⫽ 32) (n ⫽ 37) Age (years) 56 ⫾ 3 58 ⫾ 4 61 ⫾ 2 61 ⫾ 2 Height (m) 1.52 ⫾ 0.02 1.53 ⫾ 0.02 1.65 ⫾ 0.01 1.63 ⫾ 0.01 Weight (kg) 59.4 ⫾ 3.3 54.9 ⫾ 1.5 63.8 ⫾ 1.8 63.1 ⫾ 1.3 BMI (kg 䡠 m ⫺2) 25.4 ⫾ 1.1 23.4 ⫾ 0.4 23.3 ⫾ 0.6 23.7 ⫾ 0.5 Note. OSL, ossification of spinal ligaments. BMI, body mass index. Figures represent mean ⫾ SD.
consisted of 30 seconds denaturation at 94°C, 30 seconds annealing at 55°C, and 30 seconds extension at 72°C. After completing 35 cycles, an additional 5-min extension at 72°C was carried out in the GeneAmp PCR System Model 9600 (Perkin-Elmer Corp., Norwalk, CT). Amplified products were electrophoresed using 3% (w/v) agarose gel and stained with ethidium bromide. Effects of leptin on ligament cells. Human yellow ligaments (10 females) resected at surgery were used [17]. The ligaments were cut into pieces in ␣-minimum essential medium (␣-MEM), washed with ␣-MEM, placed in 350 cm 2 flasks containing ␣-MEM supplemented with 20% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 g/ml), and then incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2. The explants were cultured for 14 days with the medium change at the 7th day, then outgrowth cells were detached by 0.05% trypsin and 0.05% EDTA for 5 min at 37°C. The collected cells were seeded in 12-well plates containing ␣-MEM supplemented with 10% heat inactivated FBS, penicillin (100 U/ml), and streptomycin (100 g/ml), at a density of FIG. 2. Relationship between serum leptin level corrected by body mass index (BMI) and serum insulin level. (A) In the ossification of spinal ligaments (OSL) females, there was a positive correlation. (B) In the control females, there was a tendency of inverse but no significant correlation.
5 ⫻ 10 3 cells/cm 2. ␣-MEM, FBS, penicillin, streptomycin, trypsin and EDTA were all purchased from Gibco BRL (NY). When the cells reached confluent level, the medium was renewed, then the cells were stimulated with recombinant human leptin (R&D Systems, Minneapolis, MN) at 0, 1, 10, or 100 ng/ml, and incubated for 3, 6, or 9 days. The medium was changed once every 3 days. Alkaline phosphatase (AP) activities of the cell layer were assessed, and the data were standardized by referring to the protein content, as described previously [20, 21]. Procollagen type I carboxyl-terminal peptide (PICP) content released into the medium for 24 h was assessed using a radioimmunoassay kit (Chugai Pharmaceutical Co., Ltd., Tokyo) after the medium had been replaced with the serum-free medium (ASF-301, Ajinomoto Co., Tokyo), and the data were also standardized by referring to the protein content.
FIG. 1. Serum leptin levels corrected by body mass index (BMI) in the ossification of spinal ligaments (OSL) patients and controls. Only in the OSL females, the leptin levels were significantly higher than the control females.
Statistical analysis. To compare OSL patients and controls, Student’s t-test was done for age, height, body weight, BMI, and serum leptin level corrected by BMI. The relationship between the serum leptin level and the serum insulin level was analyzed using Pearson’s correlation analysis. Student’s t-test was also done for AP activity and PICP content between the cells with or without recombinant human leptin stimulation at various concentrations. All these analyses were performed with a significance of P ⬍ 0.05.
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Vol. 267, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2
Serum Leptin Level and Body Mass Index (BMI) of Two Female Patients Who Developed OSL at an Early Age and Who Had Severe Obesity, and Their Children
1 1’s daughter 2 2’s daughter Control females
Age (years)
BMI (kg 䡠 m ⫺2)
Leptin/BMI (ng 䡠 ml ⫺1/kg 䡠 m ⫺2)
40 17 46 25 58 ⫾ 4*
31.6 21.0 40.6 37.0 23.4 ⫾ 0.4*
0.769 0.729 0.611 1.386 0.220 ⫾ 0.018*
* Mean ⫾ SD.
RESULTS Characteristics of the subjects for measurements of serum leptin and insulin levels are shown in Table 1. There was no significant difference in the two groups. Serum leptin level corrected by BMI in the OSL females (mean ⫾ SD, 0.426 ⫾ 0.039 ng 䡠 ml ⫺1/kg 䡠 m ⫺2) was significantly (P ⬍ 0.01) higher than the control females (0.220 ⫾ 0.018 ng 䡠 ml ⫺1/kg 䡠 m ⫺2) (Fig. 1). On the other hand, in male subjects, serum leptin levels corrected by BMI were 0.144 ⫾ 0.013 ng 䡠 ml ⫺1/kg 䡠 m ⫺2 and 0.144 ⫾ 0.012 ng 䡠 ml ⫺1/kg 䡠 m ⫺2 for the OSL patients and controls, respectively. The levels were not significantly different between two male groups. In the OSL females, serum leptin level was positively correlated to the serum insulin level (r ⫽ 0.509, P ⬍ 0.05), while in the control females, there was a tendency of inverse but no significant (r ⫽ ⫺0.390, P ⫽ 0.09) correlation between serum levels of leptin and insulin (Fig. 2). The daughters of two female OSL patients had high serum leptin levels, though they did not have OSL (Table 2). RT-PCR analysis demonstrated the expression of leptin receptor mRNA in female spinal ligaments (Fig. 3), however AP activity and PICP content were not
FIG. 4. Effect of recombinant human leptin (rh-leptin) on the cultured female ligament cells. Alkaline phosphatase (AP) activity and procollagen type I carboxyl-terminal peptide (PICP) content of the cells stimulated with rh-leptin at 1, 10, or 100 ng/ml were compared to those with rh-leptin at 0 ng/ml. AP activity and PICP content in the cultured human ligament cells were not affected by exogenous rh-leptin.
affected by exogenous recombinant human leptin in the cultured female spinal ligament cells (Fig. 4). DISCUSSION
FIG. 3. RT-PCR findings. A single band of 271 bp (arrow) was obtained in the surgically resected female spinal ligaments. The lanes represent a 39-year-old female (A), a 61-year-old female (B), and a 66-year-old female (C). The result in human adipose tissue is also shown in lane (D) as a positive control.
The present study demonstrated that OSL females had a significantly high serum leptin level, and their leptin levels positively correlated to the serum insulin levels. In the control females, there was a tendency of inverse correlation between the serum levels of leptin and insulin, though it was not significant. It has been reported that alterations in plasma leptin concentrations might play a role in the etiology of insulin resistance [22] or cardiovascular disease [23]. Our findings suggest the involvement of leptin with the occurrence of female OSL.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The involvement of genetic factors has been suggested in OSL [1]. The daughters of 2 female patients who developed OSL at an early age and who had severe obesity as observed in the Zucker fatty (fa/fa) rats also had a high serum leptin level, though they had not developed OSL. These findings suggest that leptin is genetically involved with the pathogenesis of female OSL. Genetic disorders in the leptin system may be present only in females, and the mechanism of OSL development could be different according to gender. In the present study, the expression of leptin receptor mRNA was confirmed in the female spinal ligaments, but recombinant human leptin did not affect AP activity nor PICP content of the female ligament cells, which are markers for the bone formation [24, 25]. Another study also reported that leptin didn’t have any significant direct effect in controlling bone cell activity [26]. Leptin was reported to suppress insulin secretion [27], and insulin has been thought to accelerate bone formation [28 –30]. A previous study [2] showed that OSL patients have high serum insulin levels. Therefore, leptin may affect indirectly the occurrence and establishment of female OSL when leptin is not able to suppress insulin because of a certain genetic abnormality. On the other hand, leptin was reported to act on human marrow stromal cells to enhance differentiation to osteoblasts [31], and leptin may also have direct effects on heterotopic bone formation in the human spinal ligaments. The effect of leptin to bone formation is still controversial. ACKNOWLEDGMENT This work was supported by a grant-in-aid from the Investigation Committee on the Ossification of Spinal Ligament, Ministry of Health and Welfare of Japan.
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