E. STATISTICAL ANALYSIS
IV. DISCUSSION
Bone metabolism, which was influenced by hormones, growth factors, and cytokines, orchestrate the activities of both osteoclast and osteoblast cells. Bone mass is maintained through a balance between bone formation and resorption, there are three possible mechanisms by which a lower bone volume can be achieved, the first is to decrease bone formation, the second is to increase bone resorption, and the third is combination of the first and the second. OLETF rat’s body weight and body composition was significant higher than LETO rat’s body weight and body composition, but OLETF rat’s bone mineraldensity was significant lower LETO rat’s from 40wks in this study.
Sex steroids and parathyroid hormone are well known hormones in regulating bone resorption. No hormone has been shown to control bone formation. As type II DM model, male OLETF rat was developed DM in 24wks, and the function of insulin secretion was reduced from 36week. In patients with Type II DM, male hypogonadism has been reported (Fushimi H et al, 1989). Although obesity or Type II DM is closely related to gonadal dysfunctions, details of the mechanism remain unclear (Kawano K et al, 1992). It is also well known, when the serum testosterone decreased, the osteoblast cell production of transforming growth factor- ß (TGF-ß) was also decreased. TGF-ß treatment increase bone formation parameters in vitro and in vivo and decreases osteoclaste cell survival in vitro. In this study, all of the OLETF rats are developed DM in 25wks, serum testosterone also decreased in 25wks. In 40wks and 52wks, that was the similar as the former study (KATSUMORI KOMAKI et al, 2005), and it leads to increase of bone resorption. Because
hormone in this study.
Conflicting reports existed in regarding the influence of impaired insulin secretion and metabolic control on bone metabolism (McNair P et al, 1979; Munoz-Torres M et al, 1996).
In vivo, local insulin treatment to the hemicalvaria in nondiabetic mice increased osteoid volume and the number of osteoblasts (Cornish J et al, 1996). High extracellular glucose levels have been found to cause changes in osteoblastic gene expression (Hough S et al, 1981), inhibit osteoblast-like cell growth (Terada M et al, 1998) and promote osteoclastic bone resorption (Williams JP et al, 1997). From the change of OLETF rat’s insulin and fasting glucose in this study, we can infer that decreased insulin may decrease bone formation, high level of glucose may increase bone resorption.
Serum IGF-I was correlated with body composition and BMD has been confirmed in former study (H. N. Rosen et al, 1995). The IGF-I circulated in blood and interaction on epiphyseal growth plate increased unilateral bone growth significantly (J. Isgaard et al, 1986).
In this study, OLETF rat serum IGF-I was higher than LETO in the whole time before 40wks, but decreased in 40wks indicated that bone formation may also decreased from 40wks.
It is well known that Glucocoid hormones increase bone resorption. Corticosterone is the main glucocoid hormone in rat, and the high level of corticosterone decrease bone formation; in human, glucocorticoid decrease osteoblast differentiation and increase bone loss. In this study, OLETF rat’s corticosterone was significant higher than LETO rat’s from 40wks to 52wks, so corticosterone may also influence the BMD.
Exceeded thyroid hormone stimulates bone resorption, resulting in increased bone turnover and both cortical and trabecular bone loss (Meunier PJ et al, 1972; Mosekilde L and Melsen F, 1978). In this study, the free T4 have no significant difference between two groups
in 40wks, indicated that free T4 may have no influence in bone metabolism of OLETF rat.
Leptin is almost exclusively produced by fat with a very strong association between serum leptin and fat mass (Maffei M et al, 1995; Considine RV et al, 1996; Thomas T et al, 2000). Leptin’s effect on BMD is complex and likely to be mediated through direct and indirect mechanisms (Reseland JE and Gordeladze JO, 2002; Ruhl C and Everhart J, 2002;
Reid IR and Comish J, 2004). Previous study showed that as a potent inhibitor of bone formation, leptin acting through the central nervous system in mice (Ducy et al, 2000), it enhances osteoblast formation and inhibits osteoclast generation (Holloway WR et al, 2002;
Cornish J et al, 2002). Centrally, leptin has been shown to inhibit bone formation through a hypothalamic relay, an effect that is inhibited using blockers (Ducy P et al, 2000; Cock TA and Auwerx J, 2003). The leptin pathway acts both to stimulate bone formation when serum leptin levels are absent and to suppress bone formation when present in excess (Ducy, P. et al, 2000). OLETF rat serum leptin levels was significant higher than LETO rat from 8wks to 52wks, and in 8wks and in 25wks was significant higher BMD than LETO rat indicated that 8wks and 25wks serum leptin wasn’t exceeded, but from 40wks was exceeded. NPY2-R expressed strongly pattern in OLETF rat hypothalamic arcuate NPY2 was mediated with serum letpin, when leptin was higher, NPY2-R was over expressed, and it decreased bone formation. From Paul’s study (Paul A. Baldock et al, 2002), hypothalamic Y2 receptor deletion were significantly increased bone formation in mice. In this study, high level of serum leptin was observed from 8wks to 25wks indicated that serum leptin in this period was very important in evaluation of BMD. NPY2 in hypothalamic arcuate functions is also important in evaluation of BMD in post 40wks.
activator of NF-kB ligand (RANKL) regulation osteoclast specialization (Yasuda H et al, 1998). RANKL was position on membrain and combination with osteoclast progenitor receptor activator of nuclear factor kappa B (RANK), increase the osteoclast cell’s formation and activity. RANKL’s bone resorption activity was regulated with OPG, and OPG was combination with RANKL, and interrupt RANKL and RANK interaction than control inhibite osteoclast (Suda T et al, 1999; Hofbaner LC et al, 2000). From OPG level lower in OLETF rat than LETO rat in 40wks and 52wks, we found that the bone resorption in OLETF rat was more activity than LETO rat. CTx was formed depend on cathepsin K which is important enzyme of bone’s collegen resolution (Bonde M et al, 1997), because the high level of CTx was reflection excellent of resorption. OLETF rat’s CTx was higher than LETO in 40wks and 52wks in this study, both of bone resorption marker reflection that OLETF rat resorption was excellent in 40wks and 52wks than the LETO rat, it also indicated that OLETF rat’s bone resorption more activity than LETO rat from 40wks.
For the limitation of this study, body composition was measured with PIXImusII which is the model of small animal, the rat body was sectioned eight parts, and the data were combinded. All the rats were anethetized more than ten times and blood sample were collected five times, rat’s body weight was not increased significantly like other studies.
VI. CONCLUSION
OLETF rat had low bone density after 40wks of age and this finding could be correlated with low level of testosterone, high level of corticosterone and leptin in serum, and also NPY2-R over-expression in hypothalamic arcuate nuclei.
.
Fig. 1. OLETF rat’s weight was higher than LETO in whole time (P<0.001).
Fig.1. OLETF rats body weight higher than LETO in the whole time(P<0.001).
Fig. 2. OLETF rat’s body composition was higher in 25wks, 40wks and 52wks (P<0.001).
Fig. 3. OLETF rat’s femur BMD was lower than LETO (P<0.05) in 40wks and 52wks.
0
Ta b l e 1 . F a s t i n g g l u c o s e , I n s u l i n , L e p t i n , Te s t o s t e r o n e , I G F - I , f r e e T 4 , a n d C o r t i c o s t e r o n e i n 8 w k s ,
2 5 w k s , 4 0 w k s a n d 5 2 w k s .
8 w k s 2 5 w k s 4 0 w k s 5 2 w k s
L E T O O L E T F L E T O O L E T F L E T O O L E T F L E T O O L E T F ( n = 1 1 ) ( n = 1 1 ) ( n = 1 1 ) ( n = 1 1 ) ( n = 1 1 ) ( n = 1 1 ) ( n = 1 1 ) ( n = 1 1 )
G l u c o s e - - - - - - 1 2 0 . 4 6 ± 6 . 6 4 1 3 5 . 7 8 ± 7 . 7 4* 1 5 9 . 8 6 ± 8 . 5 4 2 3 2 . 9 7 ± 1 8 . 7 7* 1 3 1 . 2 7 ± 2 3 . 5 0 3 2 2 . 2 6 ± 1 3 3 . 9 6* ( m g / d l )
I n s u l i n 0 . 5 4 9 ± 0 . 1 5 4 0 . 5 8 5 ± 0 . 1 0 0 0 . 7 3 ± 0 . 2 9 1 . 9 3 1 ± 0 . 4 9 3* # 0 . 6 0 ± 0 . 1 8 7 0 . 7 6 1 ± 0 . 4 3# 0 . 7 8 0 ± 0 . 2 0 9 0 . 6 3 1 ± 0 . 1 4 6 ( n g / m l )
L e p t i n 3 . 1 2 ± 1 . 0 4 7 . 0 4 ± 1 . 5 2* 1 0 . 6 8 ± 2 . 0 5# 2 9 . 7 9 ± 7 . 7 7* # 4 . 3 1 ± 0 . 7 9# 1 1 . 9 3 ± 5 . 2 1* # 6 . 8 9 ± 0 . 8 9# 5 . 8 9 ± 2 . 6 3# ( n g / m l )
( n g / m l ) ( n g / m l ) ( n g / m l )
Te s t o s t o s t e r o n e 3 2 1 . 4 ± 1 5 1 . 2 3 4 9 . 7 ± 1 1 5 . 9 2 2 6 . 0 ± 9 4 . 1 1 7 6 . 5 ± 3 2 . 9# 2 1 8 . 7 ± 7 4 . 5 1 0 1 . 6 ± 3 6 . 5* # 1 6 6 . 7 ± 6 7 . 6 9 2 . 5 ± 1 8 . 2* ( n g / d L )
I G F - I 1 0 1 1 . 6 ± 4 4 . 3 1 2 8 8 . 4 ± 9 1 . 2* 1 0 4 3 . 5 ± 6 9 . 4 1 3 7 3 . 7 ± 1 5 5 . 7* 8 2 5 . 6 ± 3 4 . 0# 1 2 9 7 . 4 ± 1 5 8 . 4* 1 5 5 5 . 4 ± 1 4 1 . 9# 1 8 2 5 . 6 ± 1 5 5 . 9* # ( n g / m l )
f r e e T 4 1 . 2 8 4 ± 0 . 0 8 9 1 . 5 4 8 ± 0 . 1 6 5* 1 . 0 5 1 ± 0 . 1 1 3# 1 . 4 3 4 ± 0 . 0 8 7* # 1 . 0 4 8 ± 0 . 0 7 1 1 . 0 5 3 ± 0 . 0 7 9# 0 . 6 0 8 ± 0 . 0 7 3# 0 . 4 2 3 ± 0 . 0 8 2* # ( n g / d L )
C o r t i c o s t e r o n e 4 0 5 . 6 ± 4 1 . 4 2 6 2 . 2 ± 5 1 . 7* 1 9 5 . 3 ± 2 5 . 8 #2 4 1 . 4 ± 5 5 . 1* 3 5 0 . 0 ± 6 1 . 2# 4 1 5 . 9 ± 8 1 . 4# 2 0 1 . 3 ± 3 4 . 9 # 3 5 1 . 2 ± 6 3 . 3* ( n g / m l )
* : p < 0 . 0 5 c o m p a r e d t o L E T O . # : p < 0 . 0 5 c o m p a r e d t o b a s e l i n e
Fig. 4. Ob-R and NPY2-R immunostaining in LETO and OLETF brain tissues.
(LSAB, ××××200)
A, D: Increased cytoplasmic and membrane expression for Ob-R in LETO.
B, C : weekly positive cytoplasmic and membrane staining for Ob-R in OLETF.
Table 2. Expression of Ob-R in LETO (A) and OLETF (B) (P=1.000), NPY2-R in LETO (C) and OLETF (D)( P=0.017 ).
+ ++ +++
Ob-R LETO 6 (54.5%) 2 (18.1%) 3 (27.2%)
Ob-R OLETF 5 (45.5%) 4 (36.3%) 2 (18.1%)
NPY2-R LETO 6 (54.5%) 5 (45.6%) 0 (0%)
NPY2-R OLETF 3 (27.3%) 2 (18.1%) 6 (54.5%)
B BB B
C C C
C DDDD
AA AA
Fig. 5. OLETF rat’s CTx was higher than control in 40wks .
* compare to LETO in the same week(P<0.05), # compare to base line(P<0.05).
Fig. 6. OLETF rat’s OPG was lower than control in 40wks(P<0.05).
* compare to LETO in the same week(P<0.05), # compare to base line(P<0.05).
REFERENCES
1. Baskin, D.G., Breininger, J.F., and Schwartz, M.W. Leptin receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus.
Diabetes. 48:828-833, 1999
2. Bjurholm A: Neuroendocrine peptides in bone. Int Orthop.15(4):325-9,1991
3. Broberger, C., Landry, M., Wong, H et al. Subtypes Y1 and Y2 of the neuropeptide Y receptor are respectively expressed in pro-opiomelanocortin- and neuropeptide-Y-containing neurons of the rat hypothalamic arcuatee nucleus. Neuroendocrinology.
66:393-408, 1997
4. Bonde M, Garnero P, Fledelius C et al. Measurement of bone degradation products in serum using antibodies reactive with an isomerized form of an 8 aminoacid sequence of C-telopeptide of type 1 collagen. J Bone Miner Res 12(7):1028-34, 1997
5. Christensen JO, Svendsen OL. Bone mineral in pre- and postmenopausal women with insulin-dependent and non-insulindependent diabetes mellitus. Osteoporos Int 10:307–
311, 1999
6. Cinti S, Frederich RC, Zingaretti MC et al. Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue. Endocrinology 138:797-804, 1997
7. Cock TA, Auwerx J. Leptin: cutting the fat off the bone. Lancet 362:1572–1574, 2003
8. Considine RV, Sinha MK, Heiman ML et al. Serum immunoreactive- leptin concentrations in normal-weight and obese humans. N Engl J Med 334:292–29, 1996
9. Cornish J, Callon KE, Reid IR. Insulin increases histomorphometric indices of bone formation in vivo. Calcif Tissue Int 59:492–5, 1996
10. Cornish J, Callon KE, Bava U et al. Leptin directly regulates bone cell function in vitro and reduces bone fragility in vivo. J Endocrinol 175:405–415, 2002
11. Ducy, P., Amling, M., Takeda, S. et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100:197-207, 2000
12. El Miedany YM, el Gaafary S, el Baddini MA. Osteoporosis in older adults with non-insulin-dependent diabetes mellitus: Is it sex related? Clin Exp Rheumatol 17:561–567, 1999
13. Felson DT, Zhang Y, Hannan MT et al. Effects of weight and body mass index on bone mineral density in men and women: the Framingham study. J Bone Miner Res 8:567-573, 1993
14. Friendman JM, Halaas JL. Leptin and the regulation of the body weight in mammals.
Nature (Lond) 395:763-770, 1998
15. Fushimi H, Hovie H, Inoue T et al. Low testosterone levels in diabetic men and anmals: a possible role in testicular importance. Diabet Res Clin Pract 6: 297-301, 1989
16. Golden PL. Maccagnan TJ, Pardridge WM: Human blood-brain barrier leptin receptor.
Binding and endocytosis in isolated human brain microvessels. J Clin Invest 99:14-18, 1997
17. Goodman WG, Hori MT. Diminished bone formation in experimental diabetes.
Relationship to osteoid maturation and mineralization. Diabetes 33:825–831, 1984
18. Herzog H: Hypothalamic Y2 Receptors: Central Coordination of Energy Homeostasis and Bone Mass .Regulation.Drug News Perspect. 15(8):506-510, 2002
19. H. N. Rosen, V. Chen, A. Cittadini et al. Treatment with growth hormone and IGF-I in growing rats increases bone mineral content but not bone mineral density. J.Bone Miner, Res 10: 1352-1358, 1995
20. Hofbaner LC, Khosla S, Dunstan CR et al. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 15:2-12, 2000
21. Holloway WR, Collier FM, Aitken CJ et al. Leptin inhibits osteoclast generation. J Bone Miner Res 17:200–209, 2002
22. Hough S, Avioli LV, Bergfeld MA et al. Correction of abnormal bone and mineral metabolism in chronic streptozotocin-induced diabetes mellitus in the rat by insulin therapy. Endocrinology 108:2228–34, 1981
23. James F. Whitfield, PhD, Leptin—A New Member of the Bone Builders’ Club? FRSC.
24. J Cornish, K E Callon, U Bava et al. Leptin directly regulates bone cell function in vitro and reduces bone fragility in vivo Journal of Endocrinology 175:405–415, 2002
25. J. Isgaard, A. Nilsson, A. Lindahl et al. Effects of local administration of GH and IGF-I on longitudinal bone growth in rats. Am. J. Physio. 250:E367-E372, 1986
26. Kawano K, Hirashima T, Mori S et al. OLETF(Otsuka Long Evans Tokushima Fatty) rat;
a new NIDDM rat strain. Diab Res Clin Pract 24:317-320, 1994
27. Kawano K, Hirashima T, Mori S et al. Spontaneous long-term hyperglycemic rat with diabetic complications. Diabetes 41:1422-1428, 1992
28. Kawano K, Hirashima T, Mori S et al. OLETF(Otsuka Long Evans Tokushima Fatty) rat; a new NIDDM rat strain. Diab Res Clin Pract 24:317-320, 1994
29. Kawano K, Hirashima T, Mori S et al. Spontaneously diabetic rat “OLETF” as a model for NIDDM in humans. In Frontiers in animal Diabetic Reserch. Lesson from Animal Diabetes II: ed. Shafrir E Boston Birkhauser :225-236,1996
30. KATSUMORI KOMAKI, YASUHIRO OHNO and NORIHIKO AOKI. Gonadal hormones and gonadal function in type 2 diabetes model OLETF(Otsuka Long Evans Tokushiwa Fatty)Rats. Endocrine Journal 52(3):345-351, 2005
31. Kazuya Kawano, Tsukasa Hirashima, Shigehito Mori et al. Establishment of the OLETF rat. Obesity and NIDDM. Lessons from the OLETF Rat. 1-11, 1999
32. King, P.J., Williams, G., Doods, H et al. Effect of a selective neuropeptide Y Y(2) receptor antagonist, BIIE0246 on neuropeptide Y release. Eur. J. Pharmacol. 396:R1- R3, 2000
33. Leidig-Bruckner G, Ziegler R. Diabetes mellitus a risk for osteoporosis? Exp Clin Endocrinol Diabetes 109(Suppl 2):S493–514, 2001
34. Maffei M, Halaas J, Ravussin E et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1:1155–
1161, 1995
35. Marie-Louise Håkansson, Hilary Brown, Nico Ghilardi et al. Leptin Receptor Immunoreactivity in Chemically Defined Target Neurons of the Hypothalamus.The Journal of Neuroscience. 18(1):559–572, 1998
36. McNair P, Madsbad S, Christiansen C et al. Bone loss in diabetes: effects of metabolic state. Diabetologia 17:283–6, 1979
37. Melanie Henry, Lorraine Ghibaudi, Jun Gao et al. Hwa: Energy Metabolic Profile of Mice after Chronic Activation of Central NPY Y1, Y2, or Y5 Receptors. OBESITY RESEARCH 13(1):36-47, 2005
38. Meunier PJ, Bianchi GCS, Edouard CM et al. Bony manifestations of thyrotoxicosis.
Orthop ClinNorth Am 3:745–74, 1972
39. Mosekilde L, Melsen F. Effect of antithyroid treatment on calcium–phosphorus metabolism in hyperthyroidism. III. Bone histomorphometry. Acta Endocrino87:751–8, 1978
40. Munoz-Torres M, Jodar E, Escobar-Jimenez F et al. Bone mineral density measured by dual X-ray absorptiometry in Spanish patients with insulin-dependent diabetes mellitus.
Calcif Tissue Int 58:316–9, 1996
41. PABLO BRUMOVSKY, DAVOR STANIC, SAM SHUSTER et al. .Neuropeptide Y2 Receptor Protein Is Present in Peptidergic and Nonpeptidergic Primary Sensory Neurons of the Mouse. THE JOURNAL OF COMPARATIVE NEUROLOGY 489:328–
348, 2005
42. Paul A Baldock, Amanda Sainsbury, Susan Allison et al. Hypothalamic Control of Bone Formation: Distinct Actions of Leptin and Y2 Receptor Pathways:JOURNAL OF BONE AND MINERAL RESEARCH 20(10):1851-1857, 2005
43. Reid IR, Comish J. Direct actions of leptin on bone remodeling. Calcif Tissue Int 74:313–316,2004
44. Reseland JE, Gordeladze JO. Role of leptin in bone growth: central player or peripheral supporter? FEBS Lett 528:40–42, 2002
46. Robert S. Weinstein, Robert L. Jilka, A. Michael Parfitt, and Stavros C. Manolagas.
Inhibition of Osteoblastogenesis and Promotion of Apoptosis of Osteoblasts andOsteocytes by Glucocorticoids. J. Clin. Invest 102: 274-282, 1998
47. Ruhl C, Everhart J. Relationship of serum leptin concentration with bone mineral density in the United States population. J Bone Miner Res 17:1896–1903, 2002
48. SHENG BI, ELLEN E. LADENHEIM, GARY J. SCHWARTZ et al. MORAN :role for NPY overexpression in the dorsomedial hypothalamus in hyperphagia and obesity of OLETF rats :Am J Physiol Regulatory Integrative Comp Physiol. 281:R254-R260, 2001
49. Shires R, Teitelbaum SL, Bergfeld MA et al.The effect of streptozotocin-induced chronic diabetes mellitus on bone and mineral homeostasis in the rat. J Lab Clin Med 97:231–240, 1981
50. SHU TAKEDA and GERARD KARSENTY. Central control of bone formation. J Bone Miner Metab 19: 195-198, 2001
51. Stephens, T.W. et al.. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 377:530-532, 1995
52. Suda T, Takahashi N, Udagawa N et al. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocrine Rev 20: 345-357, 1999
53. Terada M, Inaba M, Yano Y et al. Growth-inhibitory effect of a high glucose concentration on osteoblast-like cells. Bone 22:17–23, 1998
54. Thomas T, Burguera B, Melton III LJ et al. Relationship of serum leptin levels with body composition, sex steroid, and insulin levels in men and women. Metabolism 49:1278–1284, 2000
55. Ulrika Smedh,,CA Marie-Louise Håkansson, Björn Meister and Kersten Uvnäs Moberg. Leptin injected into the fourth ventricle inhibits gastric emptying .NeuroReport 9:297–301, 1998
56. Weinstock RS, Goland RS, Shane E et al. Bone mineral density in women with type II diabetes mellitus. J Bone Miner Res 4:97–101, 1989
57. Williams JP, Blair HC, McDonald JM et al. Regulation of osteoclastic bone resorption by glucose. Biochem Biophys Res Commun 235:646–51, 1997
58. Yasuda H, Shima N, Nakagawa N et al. osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANK. Proc Natl Acad Sci U.S.A 95:3597-3602, 1998
59. Yun-Jung Leea, Jung-Hyun Parkb, Sung-Kyu Juc et al. _Leptin receptor isoform expression in rat osteoblasts and their functional analysis :FEBS Letters 528 :43-47, 2002
-국문요약국문요약국문요약국문요약-
결론 결론결론
결론: OLETF쥐에서 40주부터 골밀도가 낮게 나타난 것은 혈액내의 고농도의 렙틴, 그리 고 hypothalamic arcuate에서 NPY2수용체의 과발현과 연관이 있으며, 40주부터 성선기능이 저하되고 코르티코스테론이 과분비되어 혈액내의 렙틴과 hypothalamic arcuate의 NPY2로 인한 골흡수 증가를 감당할 수 없기 때문으로 생각된다.
핵심되는 핵심되는핵심되는
핵심되는 말말말: 골대사, NPY2 수용체, 렙틴, arcuate, hypothalamic, 호르몬 말