NUT9158_proof ■ 31 December 2013 ■ 1/7

Nutrition xxx (2014) 1–7

Contents lists available at ScienceDirect

Nutrition
journal homepage: www.nutritionjrnl.com

1
2
3
4
5
6
Q3
7
8 Q1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52

Original article

Shark protein improves bone mineral density in ovariectomized
rats and inhibits osteoclast differentiation
Kazuki Uehara M.S. a, Akira Takahashi b, Mutsuto Watanabe M.S. a,
Yoshihiro Nomura Ph.D. a, *abApplied Protein Chemistry, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan
Chuka Takahashi Corporation, Chuo-ku, Tokyo, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 15 April 2013
Accepted 16 November 2013

Objectives: Fish proteins are potential sources of natural drugs and food additives. There are manystudies being performed to develop underutilized ﬁsh proteins. Therefore, the aim of this study
was to determine how shark protein functions as a dietary supplement for bone health.
Methods: Three groups of ovariectomized (OVX) rats were fed different diets containing 20% casein
protein, 20% shark protein, or 20% cod protein for 4 wk. Bone mineral density of the right femur
was measured by dual-energy x-ray absorptiometry and quantitative computed tomography.
Furthermore, we prepared low-molecular-weight peptides from shark protein using protease forin vitro studies. Calcitriol was added to bone marrow cells and the receptor activator of the nuclear
factor-kB ligand was added to RAW264 cells. After 7 d, the number of tartrate-resistant acidphosphatase-positive cells was counted.
Results: In the shark protein-fed group, bone mineral density of the femur epiphysis was higher
than that of the casein protein-fed group. In particular, the shark protein-fed group showed anincrease in bone mineral density of that was represented mainly by trabecular bone. Shark proteinhydrolysates inhibited osteoclast formation in bone marrow cells and RAW264 cells.
Conclusions: These results suggest that shark protein might suppress the bone loss caused by estrogen deﬁciency through the suppression of osteoclast formation.
! 2014 Elsevier Inc. All rights reserved.

Keywords:
Postmenopausal osteoporosis
Ovariectomized rats
Osteoclast differentiation
Shark protein
Bone resorption

Introduction
Shark protein, which is available on the world market, usuallycomes from sharks that are caught during tuna trawler ﬁshing.
The commercial value of the shark is represented mainly by its
ﬁn, which is used as a luxury foodstuff. Therefore, shark ﬁns arenormally obtained by the controversial practice of ﬁnning, inwhich a shark’s ﬁn is cut off and the rest of its body is tossed into
the ocean. However, in Japan, the entire shark body is utilized. Its
cartilage is used as a source of chondroitin sulfate and collagen is
puriﬁed from its skin. Although shark protein is the main
ingredient in ﬁsh-paste products, its commercial value remains
low. In our laboratory, we examined how shark protein can beused to increase its commercial value.

KU performed the experiment and drafted the manuscript. AT and MW advisedon the experimental design. YN offered instruction and advice on the
manuscript.
* Corresponding author: Tel.: þ81 42 367 5790; fax: þ81 42 367 5791.
E-mail address: Ny318@cc.tuat.ac.jp (Y. Nomura).

Bone is an important organ that regulates mineral homeostasis. Menopause causes physiological changes that can lead toan imbalance between bone formation and bone resorption,
resulting in net bone loss and osteoporosis, mainly causedby estrogen deﬁciency [1,2]. Current therapies for osteoporosis
include estrogen replacement therapies and the use of bisphosphonates. These therapies are effective in preventing boneloss caused by menopause, but some are accompanied byadverse side effects, such as uterine bleeding, carcinogenesis,
and cardiovascular disease [3–5]. Therefore, diet therapy andlifestyle changes that minimize bone loss in postmenopausal
women would be very helpful in decreasing the need for drug
therapy to prevent osteoporosis. Recently, hydrolyzed collagen of
porcine origin [6], oil palm leaf extract [7], soy isoﬂavones plus
vitamin D3 [8], and lactoferrin [9] have become available as foodsupplements for improving bone mineral density (BMD). However, few studies have examined ﬁsh protein–derived foodfactors that have a beneﬁcial effect on BMD. Therefore, the aimof this study was to determine how shark protein functions as
a dietary supplement for bone health.

0899-9007/$ - see front matter ! 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nut.2013.11.005

Please cite this article in press as: Uehara K, et al., Shark protein improves bone mineral density in ovariectomized rats and inhibits
osteoclast differentiation, Nutrition (2014), http://dx.doi.org/10.1016/j.nut.2013.11.005

53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105

NUT9158_proof ■ 31 December 2013 ■ 2/7

2

106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169

K. Uehara et al. / Nutrition xxx (2014) 1–7

Materials and methods

Table 2
Amino acid concentrations in casein, shark protein, and cod protein

Materials
Seventeen-wk-old female Wistar rats were purchased from the Sankyo Labo
Service Corporation, Inc. (Tokyo, Japan). Casein was purchased from the Oriental
Yeast Co., Ltd. (Tokyo, Japan). Alcalase"2.4 L FG was purchased from Novozymes
A/S (Bagsvaerd, Denmark). Umamizyme G was purchased from Amano Enzyme
Inc. (Aichi, Japan). Murine macrophage RAW 264 cells were provided by RIKEN
BRC through the National Bio-Resource Project of the MEXT (Ibaraki, Japan). Fetal
bovine serum (FBS), penicillin–streptomycin–neomycin (PSN) antibiotic mixture(100 X), non-essential amino acids, and minimum essential medium (MEM) werepurchased from Life Technologies Japan Ltd. (Tokyo, Japan). Receptor activator of
nuclear factor-kB ligand from mouse (RANKL), 17 b-estradiol (E2), calcitriol(1 a,25-dihydroxy vitamin D3 [1,25(OH)2 D3]), and a-MEM were purchased from
Sigma-Aldrich Inc. (St. Louis, MO, USA). Tartrate-resistant acid phosphatase(TRAP) staining kit was purchased from Primary Cell Co., Ltd. (Hokkaido, Japan).
Other chemical reagents were all of special grade from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan)
Preparation of shark protein hydrolysates
Shark protein hydrolysates (SH) were made from fresh great blue shark(Prionace glauca) protein. Shark protein was heated in 80" C water for 3 min, andthen compressed to remove excess water. The same quantity of distilled water
was added along with proteases (Alcalase"2.4 L FG) at 3.333% (dry weight). After
30 min, the protein was further treated with 0.5% (dry weight) proteases(umamizyme G) at 50" C and pH 7.0 for 24 h. Alcalase"2.4 L FG is an exo-type
peptidase and umamizyme G is a mixture of endo- and exo-type peptidases
with a broad substrate speciﬁcity. The reaction was stopped by heating at 85" Cfor 20 min. After all hydrolysates were centrifuged to remove the precipitates, thesupernatant was collected and freeze-dried. To compare with SH, cod proteinhydrolysates (CH) also were prepared in the same manner.
Amino acid analysis of casein, shark protein, and cod protein using
high-performance liquid chromatography
To determine the amino acid proﬁle of casein, shark protein, and cod protein,reverse-phase chromatography was used. Amino acid analysis was performed
using a previously described method [10], with slight modiﬁcations [11]. The
resulting phenylthiocarbamyl amino acids were separated using an octadecylsilane column (TSKgel ODS-80 TsQA 4.6 mm # 150 mm; Tosoh Corp., Tokyo,
Japan) at 1.0 mL/min using a binary linear multistep solvent gradient. Solution A
consisted of 50 mM sodium acetate buffer, pH 6.0, containing 3% acetonitrile.
Solution B consisted of 60% acetonitrile. Gradient proﬁle was: 0 to 15 min, 0 to
70% B; 15 to 25 min, 70 to 100% B; 25 to 26 min, 100% B; 26 to 28 min, 0% B. The
column was maintained at 40" C. Elution peaks were monitored at 254 nm.

Amino acid weight ratio (%)

CaseinShark protein

Cod protein

Asparitic acid/asparagine
Glutamic acid/glutamine
Hydroxyproline
SerineGlycineHistidine
Arginine
Threonine
AlanineProlineTyrosine
ValineMethonine
Cysteine
Isoleucine
LeucineHydroxylysine
Phenylalanine
Lysine

8.2
24.3
0.0
4.6
1.8
3.2
3.7
4.3
3.1
6.8
5.3
7.0
1.8
0.2
4.5
8.7
0.0
5.1
7.5

10.4
16.3
0.3
3.6
5.4
2.9
7.0
5.2
7.8
2.8
3.0
5.4
3.7
0.5
5.3
9.0
0.0
4.1
7.2

11.9
17.4
0.2
4.5
4.5
2.4
6.8
5.1
6.5
3.0
3.9
5.1
3.7
0.3
4.0
7.7
0.0
3.8
9.2

solutions were ﬁltered and 50 ml injected into a silica-based column (TSKgel
G2500 PWXL 7.8 mm # 30 mm; Tosoh Corp.) using a high-performance liquid
chromatography LC-8020 Model II chromatograph (Tosoh Corp.) and elution was
performed with 45% acetonitrile in water in the presence of 0.1% triﬂuoroacetic
acid at a ﬂow rate of 0.5 mL/min. The column was maintained at 40" C. The elution
peaks were monitored at 215 nm.
Animals and diets
The study was conducted in accordance with the guidelines of the Committee
for Animal Research at Tokyo University of Agriculture and Technology.
Seventeen-wk-old female Wistar rats (N ¼ 28) were given a modiﬁed AIN-93 G
diet containing 20% casein for 3 d of acclimatization ad libitum. The rats werehoused in individual cages at 22 % 1" C, 50 % 5% humidity, on a 12-h light–12-h
dark cycle, with water ad libitum. On day 7 of the experiment, 22 rats were
ovariectomized (OVX) and 6 rats were sham-operated (sham). OVX rats werethen divided into three groups of six or eight rats. They were fed ad libitum for
4 wk on a diet consisting of AIN-93 G with 200 g/kg casein protein as the protein
source; the casein being replaced with shark protein (OVX shark) or cod protein(OVX cod); the composition of this diet is presented in Table 1. Weight wasrecorded every 4 d, and food intake measured every second day of the experiment. After sacriﬁce, the right femur was excised from each rat to determinebone density after all muscles and connective tissues had been removed.

Molecular weight distribution of SH and CH by high-performance liquid
chromatography

Measurement of BMD dual-energy X-ray absorptiometry

The average-molecular-weight distributions of SH and CH were analyzed by
gel-ﬁltration chromatography. Ten mg of two types of peptide sample weredissolved in a mixture of 500 ml of ultrapure water and 500 ml of 45% acetonitrile
in water, in the presence of 0.1% triﬂuoroacetic acid. After solubilization, theThe right femur was dissected and preserved in 70% ethanol at 4" C. BMD was
measured by dual-energy x-ray absorptiometry with a Dichrom Scan PCS-600
instrument (Hitachi Aloka Medical, Ltd., Tokyo, Japan), starting scans in themost proximal area and ending in the most distal. During data analysis, the femur
was divided into 20 equal segments along its major axis [12,13].

Table 1
Composition of the diets*

CaseinShark meat
Cod meata-cornstarchb-cornstarch
SucroseSoybean oil
Cellulose powder
Mineral mix (93G-MX)
Vitamin mix (93-VX)
Choline bitartratetert-Buthylhydroquinone
Total (%)

Caseiny

Sharkz

Codx

20
0
0
13.2
40.0486
10
7
5
3.5
1
0.25
0.0014
100

0
20
0
13.2
40.0486
10
7
5
3.5
1
0.25
0.0014
100

0
0
20
13.2
40.0486
10
7
5
3.5
1
0.25
0.0014100

Animals were fed a modiﬁed AIN-93 G diet.
20% casein diet.
z
20% shark protein diet.
x
20% cod protein diet.
*yFig. 1. Elution pattern of shark protein hydrolysates (SH) and cod protein hydrolysates (CH) by gel-ﬁltration chromatography. SH prepared from shark protein and
CH prepared from cod protein were analyzed using a silica-based column. Elution
was monitored by absorbance at 215 nm. Arrows indicate elution positions ofstandard molecular weight.

Please cite this article in press as: Uehara K, et al., Shark protein improves bone mineral density in ovariectomized rats and inhibits
osteoclast differentiation, Nutrition (2014), http://dx.doi.org/10.1016/j.nut.2013.11.005

170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233

NUT9158_proof ■ 31 December 2013 ■ 3/7

K. Uehara et al. / Nutrition xxx (2014) 1–7

234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296 Q 2297

Table 3
Effect of shark protein and cod protein on several parameters*
Sham

OVX

Casein(n ¼ 6)

Casein(n ¼ 8)Shark(n ¼ 6)Cod(n ¼ 8)

Final body weight 262.3 % 9.3y 295.4 % 23.1 295.4 % 20.0 291.8 % 15.3
(g)44.9 % 8.2
42.6 % 12.6 41.3 % 8.3
Body weight gain
19.9 % 5.9y(g)y17.1 % 1.4
17.0 % 1.2
17.0 % 1.3
Food intake (g/d)
13.9 % 1.0
Uterine weight
0.66 % 0.11y 0.13 % 0.04 0.13 % 0.02 0.12 % 0.09(g)
Femur length
35.67 % 0.35 35.92 % 0.99 35.87 % 0.59 35.97 % 0.69(mm)
Femur weight (g)0.77 % 0.01
0.73 % 0.05 0.76 % 0.05 0.75 % 0.02
OVX, ovariectomized rats; Sham, sham-operated rats
* Data are shown as means % SD (n ¼ 6 or 8).
ySigniﬁcant difference from OVX casein group at P < 0.01.

3

1,25(OH)2 D3 (10-8 M), E2 (10-8 M), or SH (10 mg/mL) was added for 6 d. All
cultures were maintained at 37" C in a humidiﬁed atmosphere containing 5%
carbon dioxide in air. One-half of the medium was replaced every second d.
Murine RAW264 cells were grown in MEM supplemented with 10% FBS, 1%
PSN antibiotic mixture, and 0.1 mM non-essential amino acids. For osteoclastdifferentiation, RAW264 cells (8 # 102) in 0.2 mL were seeded into wells of a 96well plate in a-MEM supplemented with 10% FBS and 1% PSN antibiotic mixture,and cultured overnight. They were then further incubated for 7 d in the presenceof RANKL (50 ng/mL) and E2 (10-8 M), with SH (10 mg/mL) or CH (10 mg/mL). All
cultures were maintained at 37" C in a humidiﬁed atmosphere containing 5%
carbon dioxide in air. One-half of the medium was replaced every second d.
Osteoclast formation was evaluated by counting the number of TRAP-positive
stained cells.
Osteoclast differentiation assay
Osteoclast formation was measured by quantifying the cells positively stainedby TRAP. Brieﬂy, the cells were ﬁxed using 10% formalin neutral buffer solution for
5 min and then stained using a TRAP staining kit according to the manufacturer’s
instructions. TRAP-positive staining cells with more than three nuclei werecounted as TRAP-positive staining multinucleated cells using light microscopy.

Measurement of BMD by quantitative computed tomography
Statistical analyses
Total BMD, cortical BMD, and trabecular BMD of the right femur were
measured by quantitative computed tomography (QCT) using a LaTheta LCT-100(Hitachi Aloka Medical, Ltd.) with cross-sectional images of 480 # 480 pixels anda slice thickness of 1 mm. The tube voltage of the x-ray generator was 50 kV (1mA). BMD was evaluated for the proximal, middle, and distal parts of the femur
by dividing the femur into three equal lengths. The scans were analyzed with
LaTheta software (version 1.40).
Cell culture
Femoral bone marrow cells were collected from female Wistar rats (weight:
About 240 g). Femurs were removed and dissected free of adhering tissues. Boneends were removed and marrow cavities ﬂushed by slowly injecting media at one
end using a 23-gauge needle. Bone marrow cells (1.0 # 106 cells/mL) were
cultured in a-MEM containing 10% FBS and 1% PSN antibiotic mixture in 48-wellplates. The culture volume was 500 mmL per well. In addition to the presence ofData were expressed as means and SD for the number of measurements
shown in the ﬁgures or tables. Statistical analysis was carried out by analysis ofvariance, using Excel 2007 (Microsoft, Redmond, WA, USA) with the add-in
software Statcel 3 (OMS Publishing Inc, Saitama, Japan). The signiﬁcance of differences was determined using Dunnett’s multiple comparison test. The probability level used to determine statistical signiﬁcance was P < 0.05.

Results
Properties of casein, shark protein, and cod protein
Casein, shark protein, and cod protein were analyzed foramino acid composition, as shown in Table 2. Shark protein wasFig. 2. Femur mineral density by dual-energy x-ray absorptiometry (DXA). Bone mineral density (BMD) levels in femurs of sham and ovariectomized (OVX) rats fed casein,
shark, or cod diets. BMD was measured by DXA, and the femur was divided from top to bottom into 20 slices. Data for slices 3 and 18 are shown. (A) Proximal metaphysis(slice 3). (B) Distal metaphysis (slice 18). Data were shown as means % SD (n ¼ 6 or 8). *Signiﬁcant difference from OVX casein group at P < 0.05. ySigniﬁcant difference fromOVX casein group at P < 0.01.

Please cite this article in press as: Uehara K, et al., Shark protein improves bone mineral density in ovariectomized rats and inhibits
osteoclast differentiation, Nutrition (2014), http://dx.doi.org/10.1016/j.nut.2013.11.005

298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361

NUT9158_proof ■ 31 December 2013 ■ 4/7

4

362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425

K. Uehara et al. / Nutrition xxx (2014) 1–7

Fig. 3. Representative features of quantitative computed tomography images of cross sections of (A) proximal metaphysis and (B) distal metaphysis of femur. Femur
specimens were analyzed using cross-sections taken 6 mm from the proximal end and 5 mm from the distal end. The arrow in (A) indicates cortical bone area. The arrow in(B) indicates trabecular bone area. Morphometric parameters of the proximal and distal metaphysis of femurs are described in Table 4. Bars 1 mm.

obviously different from casein because it contains lower levelsof the amino acids glutamic acid/glutamine, proline, tyrosine,and valine, and higher levels of glycine, arginine, alanine, andmethionine. Furthermore, shark protein was characterized as
containing relatively high levels of isoleucine and leucinecompared with cod protein.
Properties of SH and CH
Molecular-weight distributions of SH and CH were analyzed
by gel ﬁltration chromatography, as shown in Figure 1. The
average molecular weights of SH and CH were 221 and 314,
respectively.
Effect of shark protein on the basic parameters and BMD
The rat experimental data are shown in Table 3. Although
the ﬁnal weight and food intake increased signiﬁcantly, theuterine weight decreased signiﬁcantly in the OVX casein groupcompared with the sham casein group. However, there was no
signiﬁcant difference between OVX groups in ﬁnal weight, foodintake, uterine weight, femoral bone weight, and bone length.
To examine whether the effects of shark protein and codprotein differed between trabecular and cortical bone, the effectsof these diets on BMD were compared in different slices along
the femur. Figures 2A and 2B show the BMD values of slice
numbers 3 and 18, respectively, from the experimental rat rightfemurs. The OVX shark group showed an increase in BMD at theepiphysis that was composed mainly of trabecular bone. The
BMD of the OVX shark group was higher than that of the OVX
casein group.
Representative pictures of QCT images of the proximal metaphysis and the distal metaphysis of the femur are shown in
Figure 3. Decreases in cortical bone were seen in the proximal
femur (Fig. 3A) of the OVX casein group compared with those of
the sham casein group. The OVX shark group showed increases
in cortical bone in the proximal femur similar to the sham caseingroup. Furthermore, decreases in trabecular bone were seen in

Please cite this article in press as: Uehara K, et al., Shark protein improves bone mineral density in ovariectomized rats and inhibits
osteoclast differentiation, Nutrition (2014), http://dx.doi.org/10.1016/j.nut.2013.11.005

426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489

NUT9158_proof ■ 31 December 2013 ■ 5/7

5

K. Uehara et al. / Nutrition xxx (2014) 1–7

490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553

Table 4
Quantitative computed tomography measurements of femurs in rats fed casein, shark, or cod diets*

Whole
Total BMD (mg/cm3)
Cortical BMD (mg/cm3)
Trabecular BMD (mg/cm3)
Proximal
Total BMD (mg/cm3)
Cortical BMD (mg/cm3)
Trabecular BMD (mg/cm3)
Central
Total BMD (mg/cm3)
Cortical BMD (mg/cm3)
Trabecular BMD (mg/cm3)
DistalTotal BMD (mg/cm3)
Cortical BMD (mg/cm3)
Trabecular BMD (mg/cm3)

Sham

OVX

Casein (n ¼ 6)

Casein (n ¼ 8)

Shark (n ¼ 6)

Cod (n ¼ 8)779.2 % 22.8y
1111.8 % 5.6y
489.4 % 25.4y

683.0 % 34.4
1070.2 % 24.8
402.5 % 20.9

708.0 % 21.0
1084.8 % 13.8
423.0 % 15.9

703.7 % 13.6
1083.6 % 13.5
416.2 % 13.6

843.4 % 30.0y
1089.4 % 7.9y
577.4 % 31.7y

745.9 % 33.2
1038.0 % 25.2
499.5 % 25.8

776.9 % 25.4
1964.9 % 8.2z
511.6 % 27.0

753.1 % 21.8
1050.1 % 15.9
501.1. % 24.3

837.6 % 37.4y
1259.8 % 8.9z
240.0 % 15.5z

773.3 % 38.2
1235.9 % 17.5
223.1 % 6.4

798.9 % 20.2
1246.0 % 14.8
227.6 % 11.1

805.7 % 20.7
1246.7 % 13.1
227.6 % 14.3

695.3 % 21.3y995.2 % 9.8y
531.0 % 24.8y

583.4 % 37.6
940.8 % 33.1
414.4 % 26.6

602.7 % 20.9
942.4 % 21.5
444.1 % 14.4z

606.8 % 12.7
957.3 % 15.0
134.3 % 13.9

BMD, bone mineral density; OVX, ovariectomized rats; Sham, sham-operated rats
* Data are shown as means % SD (n ¼ 6 or 8).
ySigniﬁcant difference from OVX casein group at P < 0.01.
zSigniﬁcant difference from OVX casein group at P < 0.05.

the distal femur (Fig. 3B) of the OVX casein group compared withthose of the sham casein group. The OVX shark group showed
increases in trabecular bone in the distal femur to levels similar
to the sham casein group.
Total BMD, cortical BMD, and trabecular BMD of the rightfemur were measured by QCT. Total BMD, cortical BMD, andtrabecular BMD decreased signiﬁcantly in the OVX casein groupcompared with the sham casein group (Table 4). The OVX sharkgroup showed an increase in cortical BMD at the proximal sites ofthe femur. Furthermore, the OVX shark group showed an increase in trabecular BMD at the distal sites.
Effects of SH on 1,25(OH)2 D3-induced osteoclast formation
Osteoclast differentiation was estimated by TRAP-positive
multinucleated cell formation. Osteoclast formation was
induced by 1,25(OH)2 D3. As shown in Figure 4A, osteoclast
formation was hardly detected when no 1,25(OH)2 D3 wasadded. However, when 1,25(OH)2 D3 was added, osteoclast formation was seen. With the addition of SH, osteoclast formation
was inhibited. The number of differentiated osteoclasts induced
by 1,25(OH)2 D3 was 28.7 % 4.8 cells per well. The addition of SH
reduced the number of TRAP- positive multinucleated cells to
35% (Fig. 4B).
Effect of SH on RANKL-induced osteoclast formation
Although the bone marrow cultures were carried out inconditions that promoted osteoclastogenesis, it is still possiblethat the effects seen depended on the presence of other cell
types. Therefore, we repeated these studies in RAW264 cells, aclonal cell line with a preosteoclast phenotype. Inhibition of
osteoclast development was again observed with SH at 10 mg/
mL with the same efﬁcacy seen in the bone marrow cultures(Fig. 4C). This suggests that SH has a direct effect on preosteoclast
cells and does not depend on the presence of other cell types. The
number of TRAP-positive cells declined by 23% in SH-treatedcultures. The number of TRAP-positive cells also declined by
15% in CH-treated cultures.

Discussion
Results of the amino acid analyses of shark protein and codprotein, showed no signiﬁcant differences (Table 2) because they
are both derived from white ﬁsh. Because the protein is largely
muscle, it can be assumed that mainly actin and myosin weredigested. However, differences in molecular weight were foundbetween SH and CH when treated with Alcalase"2.4 L FG andumamizyme G. The sharks had been caught in tuna trawl nets,
thus they had been stored on ice for a long time. In contrast, thecod had been caught during inshore ﬁshing, so they had beenfrozen quickly after being caught. Several biochemical andenzymatic changes are triggered in ﬁsh muscle soon after death,so that the peptides lower in molecular weight [14]. Enzymes arecommonly used as food-grade additives dissolved in water andthese easy to handle materials will be useful as health foods inthe future.
The ovariectomies were deemed successful as visuallyconﬁrmed by the high degree of atrophy found in all corresponding ovaries. The greater body mass accretion of the castrated rats was in agreement with other reports [15,16] and isunderstood to result from the absence of estrogen, which regulates body fat deposition and leads to more efﬁcient food conversion, the latter characteristic being veriﬁed despite theovariectomized animals being pair-fed with sham rats [16].
Figure 2 shows the lower BMD of the epiphysis from the OVX
casein group compared with the sham casein group. The pathology of the epiphysis from the OVX casein group showed alower density of trabecular bone than the sham casein group(Fig. 3). Ovariectomy stops the secretion of estrogen and decreases BMD, especially in the trabecular bone and under the
epiphyseal plate. The OVX casein group showed symptoms ofosteoporosis. However, BMD levels in the OVX shark group werehigher than in OVX casein group. Bone formation and bone
resorption were most active in the trabecular bone area
(epiphysis). As the amino acid content of the shark protein ispractically identical to that of cod protein, the different responses
observed in both the OVX shark and OVX cod groups could only
be explained by the physiological functions likely to be linked tosome of the peptides.

Please cite this article in press as: Uehara K, et al., Shark protein improves bone mineral density in ovariectomized rats and inhibits
osteoclast differentiation, Nutrition (2014), http://dx.doi.org/10.1016/j.nut.2013.11.005

554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617

NUT9158_proof ■ 31 December 2013 ■ 6/7

6

618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681

K. Uehara et al. / Nutrition xxx (2014) 1–7

Fig. 4. Inhibition of osteoclast formation by shark protein hydrolysates (SH). (A) SH was added to bone marrow cells with 1 a,25-dihydroxyvitamin D3 (D3) in 48-well plates.
After incubation for 6 d, osteoclasts (arrows) were stained using the tartrate-resistant acid phosphatase (TRAP) method. Bars 100 mm. (B) TRAP-positive multinucleated cells
that had more than three nuclei were counted in bone marrow cells. (C) RANKL-stimulated TRAP-positive multinucleated cells in RAW 264 cells. Data were shown as means %SD (n ¼ 3). E2, 17 b-estradiol; CH, cod protein hydrolysates. *Signiﬁcant difference from D3(þ) control at P < 0.01. ySigniﬁcant difference from RANKL(þ) control at P < 0.05.
zSigniﬁcant difference from RANKL(þ) control at P < 0.01.

It is generally accepted that milk is an excellent source of
calcium and contains several proﬁtable components for calcium
resorption in the intestine, such as lactose and phosphopeptides,
formed by the proteolytic digestion of milk casein [17–19].
However earlier studies conﬁrmed that calcium resorption was
unaffected by shark protein (data not shown). The shark protein
was digested into amino acids and peptides and absorbed in the
body. This might have had a direct effect on osteoclast andosteoblast activity, thus increasing the BMD. Accordingly, we
prepared low-molecular-weight peptides from shark and codprotein using Alcalase"2.4 L FG and umamizyme G for in vitro
studies.
In bone remodeling, bone resorption by osteoclasts occurs
before bone formation by osteoblasts. Estrogen deﬁciency accelerates bone resorption, and trabecular bone becomes poriferous [20]. Therefore, suppression of bone resorption leads toarrested trabecular bone loss under estrogen deﬁciency. In thepresence of transforming growth factor-b and macrophagecolony-stimulating factor, osteoclast progenitor cells differentiate from stem cells [21–24]. In the presence of macrophagecolony-stimulating factor and RANKL, preosteoclasts differentiate from progenitor cells [25,26]. RANKL is a membrane-boundfactor that is produced by osteoblasts and stromal cells in
response to a variety of signals such as 1,25(OH)2 D3 and parathyroid hormone. RANKL acts on osteoclast progenitors to induce
osteoclast differentiation [27,28]. Although shark protein isthought to inhibit bone resorption in animals, its target cells in
relation to bone resorption and its mode of action have not been
fully elucidated. We determined the effects of SH on the function
and differentiation of osteoclasts. In this study we demonstrated
that SH inhibits TRAP-positive multinucleated cell formationinduced by 1,25(OH)2 D3 in cultures of bone marrow cells andPlease cite this article in press as: Uehara K, et al., Shark protein improves bone mineral density in ovariectomized rats and inhibits
osteoclast differentiation, Nutrition (2014), http://dx.doi.org/10.1016/j.nut.2013.11.005

682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745

NUT9158_proof ■ 31 December 2013 ■ 7/7

K. Uehara et al. / Nutrition xxx (2014) 1–7

746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802

RANKL in cultures of RAW264 cells. Our results suggest that the
suppressive effect of SH on bone resorption might mainly result
from its direct inhibitory effect on osteoclast differentiation(Fig. 4). We demonstrate the effect of SH on osteoclast differentiation and that its inhibitory effect on differentiation ispartially responsible for its suppressive effect on bone resorption
in cell culture. Similarly, in this study we demonstrated that CH
inhibits TRAP-positive multinucleated cell formation induced byRANKL in cultures of RAW264 cells. However, we included aneffective material such as shark protein, but the levels may havebeen very low because no effect was seen in vivo.
It has been reported that bioactive peptides derived frommarine species improve BMD levels in marine collagen peptides[29], and leucine–lysine–proline [30]. It has also been reported
that these peptides stimulate osteoblasts, thus increasing boneformation. However, we demonstrate here that SH acts directlyon the differentiation of osteoclasts.
Conclusion
We show that ingesting shark protein increases BMD inosteoporosis, apparently through the inhibition of osteoclast
formation. Therefore, we would like next to identify and isolate apeptide in shark protein inhibiting osteoclast differentiation.
Acknowledgments
The authors acknowledge Professor Chisato Miyaura,
Department of Biotechnology and Life Science, Tokyo Universityof Agriculture and Technology, for allowing the use of dualenergy x-ray absorptiometry; and assistant professor Maiko
Kawaguchi, Faculty of Pharmacy and Research Institute of Pharmaceutical Sciences, Musashino University, for allowing the useof quantitative computed tomography.
References[1] Richelson LS, Wahner HW, Melton LJ, Riggs BL. Relative contributions ofaging and estrogen deﬁciency to postmenopausal bone loss. N Engl J Med
1984;311:1273–5.
[2] Westerlind KC, Wronski TJ, Ritman EL, Luo ZP, An KN, Bell NH, et al. Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain. Proc Natl Acad Sci
U S A 1997;94:4199–204.
[3] Lacey JV, Mink PJ, Lubin JH, Sherman ME, Troisi R, Hartge P, et al. Menopausal hormone replacement therapy and risk of ovarian cancer. JAMA
2002;288:334–41.
[4] Trevisan M. Hormone replacement therapy and cardiovascular disease: An
evidence based approach. Nutr Metab Cardiovasc Dis 2003;13:61–3.
[5] Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, et al.
Effects of conjugated equine estrogen in postmenopausal women withhysterectomy: the Women’s Health Initiative randomized controlled trial.
JAMA 2004;291:1701–12.
[6] Guillerminet F, Fabien-Soule V, Even PC, Tome D, Benhamou CL, Roux C,
et al. Hydrolyzed collagen improves bone status and prevents bone loss inovariectomized C3 H/HeN mice. Osteoporos Int 2012;23:1909–19.
[7] Bakhsh A, Mustapha NM, Mohamed S. Catechin-rich oil palm leaf extract
enhances bone calcium content of estrogen-deﬁcient rats. Nutrition
2013;29:667–72.

7[8] Chang KL, Hu YC, Hsieh BS, Cheng HL, Hsu HW, Huang LW, et al. Combined
effect of soy isoﬂavones and vitamin D3 on bone loss in ovariectomized
rats. Nutrition 2013;29:250–7.
[9] Guo HY, Jiang L, Ibrahim SA, Zhang L, Zhang H, Zhang M, et al. Orally
administered lactoferrin preserves bone mass and microarchitecture in
ovariectomized rats. J Nutr 2009;139:958–64.
[10] Bidlingmeyer BA, Cohen SA, Tarvin TL. Rapid analysis of amino acids using
pre-column derivatization. J Chromatogr 1984;336:93–104.
[11] Sato K. Improved method for identiﬁcation and determination of ε-(gglutamyl) lysine cross-link in protein using proteolytic digestion andderivatization with phenyl isothiocyanate followed by high-performanceliquid chromatography separation. J Agric Food Chem 1992;40:806–10.
[12] Shiraishi A, Takeda S, Masaki T, Higuchi Y, Uchiyama Y, Kubodera N, et al.
Alfacalcidol inhibits bone resorption and stimulates formation in an
ovariectomized rat model of osteoporosis: distinct actions from estrogen. J
Bone Miner Res 2000;15:770–9.
[13] Yokogawa K, Miya K, Sekido T, Higashi Y, Nomura M, Fujisawa R, et al.
Selective delivery of estradiol to bone by aspartic acid oligopeptide and its
effects on ovariectomized mice. Endocrinology 2001;142:1228–33.
!mond D,[14] Bauchart C, Chambon C, Mirand PP, Savary-Auzeloux I, Re
Morzel M. Peptides in rainbow trout (Oncorhynchus mykiss) muscle subjected to ice storage and cooking. Food Chem 2007;100:1566–72.
! neo F, Amaya-Farfan J, de Assunça~o JV, Quintaes KD.
[15] de Almeida Jackix E, Cu
A food supplement of hydrolyzed collagen improves compositional andbiodynamic characteristics of vertebrae in ovariectomized rats. J Med Food
2010;13:1385–90.
[16] Hidaka S, Okamoto Y, Uchiyama S, Nakatsuma A, Hashimoto K, Ohnishi ST,
et al. Royal jelly prevents osteoporosis in rats: beneﬁcial effects in ovariectomy model and in bone tissue culture model. Evid Based Complement
Alternat Med 2006;3:339–48.
[17] Lee YS, Noguchi T, Naito H. Phosphopeptides and soluble calcium in thesmall intestine of rats given a casein diet. Br J Nutr 1980;43:457–67.
[18] Wasserman RH. Lactose-stimulated intestinal absorption of calcium: atheory. Nature 1964;201:997–9.
[19] Wong NP, Lacroix DE. Biological availability of calcium in dairy products.
Nutr Rep Int 1980;21:673–80.
[20] Parﬁtt AM, Mathews CH, Villanueva AR, Kleerekoper M, Frame B, Rao DS.
Relationships between surface, volume, and thickness of iliac trabecularbone in. J Clin Invest 1983;72:1396–409.
[21] Kodama H, Yamasaki A, Nose M, Niida S, Ohgame Y, Abe M, et al. Congenital
osteoclast deﬁciency in osteopetrotic (op/op) mice is cured by injections ofmacrophage colony-stimulating factor. J Exp Med 1991;173:269–72.
[22] Kodama H, Nose M, Niida S, Yamasaki A. Essential role of macrophagecolony-stimulating factor in the osteoclast differentiation supported bystromal cells. J Exp Med 1991;173:1291–4.
[23] Takahashi N, Udagawa N, Suda T. A new member of tumor necrosis factor
ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 1999;256:449–55.
[24] Itonaga I, Sabokbar A, Sun SG, Kudo O, Danks L, Ferguson D, et al. Transforming growth factor-beta induces osteoclast formation in the absence ofRANKL. Bone 2004;34:57–64.
[25] Quinn JM, Elliott J, Gillespie MT, Martin TJ. A combination of osteoclastdifferentiation factor and macrophage-colony stimulating factor is sufﬁcient for both human and mouse osteoclast formation in vitro. Endocrinology 1998;139:4424–7.
[26] Gyda M, Corisdeo S, Zaidi M, Troen BR. Macrophage colony-stimulating
factor suppresses osteoblast formation. Biochem Biophys Res Commun
2001;285:328–34.
[27] Fuller K, Wong B, Fox S, Choi Y, Chambers TJ. TRANCE is necessary andsufﬁcient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 1998;188:997–1001.
[28] Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Yano K, et al.
RANK is the essential signaling receptor for osteoclast differentiation factor
in osteoclastogenesis. Biochem Biophys Res Commun 1998;253:395–400.
[29] Xu Y, Han X, Li Y. Effect of marine collagen peptides on long bone development in growing rats. J Sci Food Agric 2010;90:1485–91.
[30] Huttunen MM, Pekkinen M, Ahlstrom ME, Lamberg-Allardt CJ. Effects ofbioactive peptides isoleucine-proline-proline (IPP), valine-proline-proline(VPP) and leucine-lysine-proline (LKP) on gene expression of osteoblasts
differentiated from human mesenchymal stem cells. Br J Nutr 2007;98:
780–8.

Please cite this article in press as: Uehara K, et al., Shark protein improves bone mineral density in ovariectomized rats and inhibits
osteoclast differentiation, Nutrition (2014), http://dx.doi.org/10.1016/j.nut.2013.11.005

803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859

Our reference: NUT 9158

P-authorquery-v9

AUTHOR QUERY FORM
Journal: NUT

Please e-mail or fax your responses and any corrections to:E-mail: sw.nagarajan@elsevier.com

Article Number: 9158

Fax: +91 44 4596 4899

Dear Author,
Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen
annotation in the PDF file) or compile them in a separate list. Note: if you opt to annotate the file with software other than
Adobe Reader then please also highlight the appropriate place in the PDF file. To ensure fast publication of your paper please
return your corrections within 48 hours.
For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions.
Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in
the proof.
Locationin article

Query / Remark: Click on the Q link to find the query’s location in text
Please insert your reply or correction at the corresponding line in the proof
If there are any drug dosages in your article, please verify them and indicate that you have done so byinitialing this query

Q1

Please provide academic degree for “Akira Takahashi.”

Q2

Please advise where * should appear in figure 2 or if “*Significant difference from OVX casein group at P
< 0.05” can be deleted from the legend. Footnote symbols must appear in figure.

Q3

Please confirm that given names and surnames have been identified correctly.
Please check this box or indicate
your approval if you have nocorrections to make to the PDF fileThank you for your assistance.

,