вход по аккаунту


Tetracycline administration restores osteoblast structure and function during experimental diabetes.

код для вставкиСкачать
THE ANATOMICAL RECORD 231:25-34 (1991)
Tetracycline Administration Restores
Osteoblast Structure and Function During
Experimental Diabetes
Second Department of Oral Anatomy, School of Dentistry, Showa University,
Shinagawa-ku, Tokyo, Japan (T.S., H.K.), and Department of Oral Biology and Pathology,
School of Dental Medicine, Health Sciences Center, State University of New York, Stony
Brook, New York (N.S.R., L.M.G.)
Osteopenia is a recognized complication of diabetes mellitus in
humans and experimental animals. We recently found that tetracyclines prevent
osteopenia in the streptozotocin-induced diabetic rat and that this effect was associated with a restoration of defective osteoblast morphology (Golub et al., 1990).
The present study extends these initial ultrastructural observations by assessing
osteoblast function in the untreated and tetracycline-treated diabetic rats. After a
3-week protocol, non-diabetic control and diabetic rats, including those orally administered a tetracycline, minocycline (MC), or a non-antimicrobial tetracycline
analog (CMT), were perfusion-fixed with a n aldehyde mixture; the humeri were
dissected and processed for ultracytochemical localization of alkaline phosphatase
(ALPase) and Ca-ATPase activities. Some rats from each experimental group received a n intravenous injection of 3H-proline as a radioprecursor of procollagen,
and the humeri were processed for light microscopic autoradiography. In addition,
the osteoid volume in each experimental group was quantitatively examined by
morphometric analysis of electron micrographs.
During the diabetic state, active cuboidal osteoblasts in the endosteum of control
rats were replaced by flattened bone-lining cells that contained few cytoplasmic
organelles for protein synthesis (Golgi-RER system), and active transport (mitochondria). Treating diabetic rats with MC, and even more so with CMT, appeared
to “restore” osteoblast structure. During diabetes, bone-lining cells incorporated
little 3H-proline or secreted little labeled protein and produced only a very thin
osteoid layer. Tetracycline administration to the diabetics increased both the incorporation of 3H-proline by osteoblasts and their secretion of labeled protein toward the osteoid matrix, in a pattern similar to that seen in the non-diabetic
controls. Intense alkaline phosphatase (ALPase) activity was cytochemically demonstrated along the plasma membranes of osteoblasts in the non-diabetic control
rats, but was completely absent from the bone-lining cells in the diabetics. Similar
to that described above, CMT therapy restored the ALPase activity in the diabetic
osteoblasts and the effect of MC was less dramatic. The distribution and intensity
of Ca-ATPase in the osteoblast-plasma membranes of the different groups of rats
were similar to that of ALPase, except for the absence of detectable Ca-ATPase in
the MC-treated diabetics. These results suggest that diabetes-induced osteopenia
reflects, a t least in part, impaired osteoblast structure and function and that tetracyclines, by a non-antimicrobial mechanism, may prevent this bone deficiency
by normalizing these bone-lining cells.
Osteopenia (Osteoporosis) is a complication of insulin-deficiency diabetes in humans and experimental
animals (Kaneko et al., 1990 for review). Because tetracyclines have been shown to inhibit bone resorption
induced in vitro by a variety of factors (Golub et al.,
lgS4; Rifkin et
lgS4, Comes et
we recently determined the effect Of these antibiotics (and
their non-antimicrobial analogs) on the development of
osteopenia in experimental diabetes. Of extreme inter0 1991 WILEY-LISS, INC
est, preliminary studies indicated that in vivo administration of tetracycline prevented 1) the loss of bone
density, 2) the loss of the osteoid layer, and 3) the mor-
Received June 17, 1990; accepted February 22, 1991,
Address reprint requests to Dr. T. Sasaki, Second Dept. of Oral
Anatomy, School of Dentistry, Showa University, 1-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142, Japan.
phologic degeneration of osteoblasts, all associated
with the diabetic state in rats (Golub et al., 1990).However, the mechanisms involved in ameliorating these
diabetes-induced skeletal abnormalities are as yet unknown.
Skeletal tissue metabolism is highly regulated by
bone cells such as osteoblasts and osteoclasts. Multifunctional osteoblasts synthesize and secrete organic
bone matrix consisting mainly of type I collagen
(Frank and Frank, 1969;Wright and Leblond, 1981;
Takagi et al., 1983) and regulate bone mineralization
(Fleisch and Neiman, 1961;Yoshiki et al., 1972;Fukushima and Goshi, 1983;Akisaka et al., 1988).In addition, alkaline phosphatase (ALPase) and calciumtransporting ATPase (Ca-ATPase) of osteoblasts are
thought to be involved in mineralization of bone matrix
(Fleisch and Neiman, 1961;Yoshiki et al., 1972;Jaffe,
1976; Nijweide et al., 1981; Fukushima and Goshi,
1983; Fauran-Clavel and Oustrin, 1986; Register et
al., 1986; Akisaka et al., 1988; Shen et al., 1988).
ALPase is widely recognized as a marker enzyme for
osteoblast and/or osteogenic cells (Yoshiki e t al., 1972;
Wlodarski and Reddi, 1986). Ca-ATPase participates
in the active transport of calcium ions to create local
high concentrations of this mineral at the extracellular
sites of calcification (Melancon and DeLuca, 1970;
Fukushima and Goshi, 1983;Akisaka et al., 1988).To
extend our initial studies (Golub et al., 1990) on the
prevention of osteopenia, this report describes diabetes-induced alterations in procollagen production (assessed autoradiographically) and in the cytochemical
markers of osteoblast activity (assessed ultrastructural
cytochemistry) and the response to tetracycline therapy.
Adult (4-month old) male Sprague-Dawley (Taconic
Farms, Germantown, NY) rats (350-400 g body
weight) were rendered diabetic by intravenous injection of streptozotocin (70 mg/kg body weight) as
previously described (Golub et al., 1978).Some of these
rats (4 rats per each group) were administered, by oral
gavage, minocycline (Lederle Labs, Pearl River, NY)
or 4-de-dimethylaminotetracycline (a chemically modified, non-antimicrobial analog in which the dimethylamino group on carbon-4 of the tetracycline molecule
is removed), which was synthesized and characterized
by us using techniques described previously (Golub et
al., 1987;McCormick et al., 1957;Boothe et al., 1958).
Minocycline and the chemically modified tetracycline
analog (CMT) were suspended in 2% carboxymethylcellulose and 20 mg per day was administered to the
appropriate rats throughout the 21-day protocol. The
remaining diabetic and non-diabetic (control) rats
were treated with vehicle (carboxymethylcellulose)
On day 21, the rats were fixed by intracardiac perfusion with a mixture of 1% formaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3)
for 10 min a t room temperature. After perfusion, the
humeri were dissected and immersed in the same fixative for 1 h r a t 4°C. They were then demineralized in
10% disodium ethylenediamine tetracetic acid (pH 7.3)
for 2 months at 4"C, and cross sections of 40-50 pm
thickness were prepared from mid-diaphysis of the humeri using a microslicer (Dohan EM, Osaka).
For alkaline phosphatase (ALPase) cytochemistry,
sections were prepared as above and preincubated in
0.1 M buffer containing 10 mM magnesium sulfate for
2 hr. These slices were then incubated in the following
media for 30 rnin a t room temperature (about 22°C).
The medium for ALPase, according to Mayahara e t al.
(1967),consisted of 28 mM tris-HC1 buffer (pH 8.5), 20
mM P-glycerophosphate, 3.9 mM magnesium sulfate,
2.0 mM lead citrate, and 8% sucrose. Final pH was
adjusted a t 9.0-9.2. For cytochemical controls, some
slices were incubated in a medium lacking substrate
(P-glycerophosphate) or in a complete medium containing 10 mM levamisole, a potent inhibitor of ALPase.
For calcium-transporting ATPase (Ca-ATPase) cytochemistry, the sections described above were reactivated in 0.1M sodium cacodylate buffer containing 10
mM CaC1, for 2 hr, and then incubated in the following
media for 15 min at room temperature. The medium for
Ca-ATPase, according to Ando e t al. (1981),consisted
of 3.0 mM ATP (disodium salts, Sigma Chemical), 250
mM glycine-KOH buffer (pH 9.01,10 mM CaCl,, 4.0
mM lead citrate (dissolved in 50 mM KOH), and 5 mM
levamisole. Final pH was adjusted to 9.0-9.2 using 10
M KOH. To ensure the substrate and calcium dependency of the enzymatic activity, some sections were
incubated in a medium lacking either the substrate
(ATP) or the enzyme activator CaCl,.
After incubation, these sections were postfixed with
1.5% potassium ferrocyanide-reduced 1% osmium
tetroxide for 15 min at 4°C and block-stained with ethanolated 1% uranyl acetate for 15 min. For conventional ultrathin sectioning, the bone tissues were fixed
and demineralized as described above without preparation of microslicer sections for cytochemistry. All
these specimens were dehydrated through a graded
ethanol series and embedded in Quetol 812 (Nisshin
EM, Tokyo). Thin sections were cut using a diamond
knife on a Reichert-Jung Ultracut OmU-4, and stained
with 1% tannic acid (pH 7.01,uranyl acetate, and lead
citrate for morphological observation or lightly stained
with uranyl acetate for cytochemistry. They were examined with a Hitachi H-800electron microscope at 75
For light microscopic autoradiography, the rats were
anesthetized with sodium nembutal and injected
through the right or left jugular vein with phosphateproline (New Enbuffered saline containing (2,3-3H)
gland Nuclear) in a dose of 20 mCiikg body weight. At
20 min and 4 h r after proline injection, the rats were
perfusion fixed through the left ventricle with a mixture of 2% glutaraldehyde and 2% formaldehyde in a
0.1 M sodium cacodylate buffer (pH 7.3)for 10 min at
room temperature. Dissected humeri were demineralized and embedded a s described above. Then 0.5-p.mthick sections were cut with a diamond knife, mounted
on glass microscopic slides, and dipped in Kodak NTB2 emulsion diluted 1:2 with distilled water. After exposure for 7 days a t 4"C, the autoradiographs were developed in Dektol (Kodak Ltd.) for 2 min at 17"C,
stained with 1% toluidine blue for 5 min a t 4WC, and
examined with a n Olympus BHS light microscope.
To evaluate the amount of osteoid produced by the
osteoblasts in each experimental group, the osteoid
Figs. 1-4. Ultrathin sections of osteoblasts and/or bone-lining cells in the non-diabetic control (l),
untreated diabetic (21, MC-treated diabetic (3), and CMT-treated diabetic rats (4). Tannic acid-uranyl
acetate-lead citrate staining. x 8,000.
Figs. 5-8
Golgi apparatus, RER, and mitochondria. Thus these
bone-lining cells were almost completely devoid of cytoplasmic organelles necessary for protein synthesis
Number of
Osteoid volume
osteoblasts and active transport (Fig. 2).
The bone-lining cells in the diabetic rats exhibited
(pm2)/10pm * S.D.
dramatic morphological changes in response to minocy36.8 * 6.0
cline (MC) administration (Fig. 3 ) and, even more so, in
Untreated diabetes
3.4 k 0.7
response to the chemically modified non-antimicrobial
16.0 2 11.0
MC-treated diabetes
tetracycline (CMT) (Fig. 4).In both the MC- and CMTCMT-treated diabetes
35.1 2 4.2
treated diabetic rats, the osteoid layer (very thin in the
untreated diabetic rats) was again present on the mineralized bone surfaces. The bone-lining cells in the MCvolume (pm2) per unit osseous surface (10 pm) of os- treated diabetic rats exhibited a mix of flattened cells,
teoblast was examined on the photographic prints pre- characteristic of the untreated diabetic bones, plus oval
pared at a final magnification of 4,000-10,000 x normal-looking cells (Fig. 3 ) . The osteoblasts in the
(original magnification of 2,000-5,000 x using a Pias CMT-treated diabetic group could not be distinguished
LA-525R image analyzer attached to a NEC personal morphologically from those in the control group, both
computer PC-9801 IVX. In each experimental group, groups exhibited active osteoblasts with similar cuboi22-28 osteoblasts were examined. Total examined ar- dal outline and a well-developed Golgi-RER system
eas of osteoid were 858.8 pm2 (control), 67.6 pm2 (un- (Figs. 1, 4).
treated diabetes), 462.7 pm2 (MC-treated diabetes) and
Histomorphometric Analysis of Osteoid Volume
921.6 pm2 (CMT-treated diabetes), respectively.
Quantitative analysis of osteoid volume in each exRESULTS
perimental group revealed that experimentally inOsteoblast Ultrastructure
duced diabetes resulted in a 91% reduction of osteoid
Because some of the ultrastructural features of the volume. MC treatment partially corrected the reduced
osteoblasts in the different groups were described pre- osteoid in the diabetics, whereas CMT therapy apviously (Golub et al., 19901, these are only reviewed peared to result in a complete restoration of this defect
briefly herein. In the non-diabetic control rats, the (Table 1).
mineralized bone surfaces of the periosteum of the hu3H-ProlineAutoradiography
meri were covered with a n unmineralized osteoid layer
In the non-diabetic controls, at 20 min after 3H-pro(Fig. 1).The osteoblasts showed a cuboidal and/or oval
outline with numerous cellular processes extending to- line injection, the silver grains appeared over the cell
wards the osteoid and into the bone canaliculi. The cell bodies of osteoblasts and fibroblasts in the periosteal
bodies contained a well-developed Golgi apparatus, mi- surfaces of the humeri (Fig. 5a). The greatest uptake of
tochondria, numerous cisternae of rough endoplasmic radioprecursor was seen in the osteoblasts. Young osreticulum (RER), and free polyribosomes (Fig. 1). Thus teocytes also incorporated radioprecursor but to a much
osteoblasts in the control rats showed a cellular mor- lesser extent. A few grains were detected over the osphology consistent with actively synthesizing and se- teoid matrix but were rarely observed over the mineralized bone matrix. At 4 h r after 3H-proline injection,
creting cells.
In contrast, the active-looking osteoblasts were miss- the silver grains were concentrated over the osteoid
ing and the adjacent osteoid volume was dramatically matrix facing the osteoblast layer (Fig. 5b). Some
decreased on the bone surfaces of the untreated dia- grains were also observed over the osteoblast-cell bodbetic rats (Fig. 2). Instead, the mineralized bone was ies and the superficial but not the deeper layers of the
covered with flattened bone-lining cells characterized mineralized bone matrix (Fig. 5b). In the untreated
by flattened nuclei and deficient cytoplasm poor in diabetics, few if any silver grains were observed over
the flattened bone-lining cells and bone matrix at either time period (Figs. 6a,b).
At 20 min after isotope injection, in both MC- and
CMT-treated diabetic rats, the silver grains appeared
Fig. 5a,b. Autoradiographs of osteoblasts in humeri of non-diabetic over the more normal-looking osteoblast-like cell bodcontrol rats a t 20 min (Fig. a) and 4 h r (Fig. b) after 3H-proline injection. Silver grains are seen over the osteoblasts a t 20 min (Fig. a) ies that replaced the flattened bone-lining cells (Figs.
7a, 8a). At 4 h r after injection, a moderate amount of
and over newly formed osteoidibone matrices a t 4 hr (b). x 600.
silver grains were observed over the osteoblasts and
Fig. 6a,b. Autoradiographs of bone-lining cells (arrowheads) of un- the osteoid matrix in the MC- and CMT-treated rats,
treated diabetic rats at 20 min (Fig. a) and 4 hr (Fig. b) after isotope
but the number of grains was fewer than that in the
injection. Note the absence of silver grains over the tissues. X 600.
non-diabetic controls (Figs. 7b, 8b).
TABLE 1. Quantitative analysis of osteoid volume per
unit osseous surface (10 am) of osteoblasts
Fig. 7a,b. Autoradiographs of osteoblasts (arrowheads) in minocycline (MCbtreated diabetic rats at 20 min (Fig. a) and 4 hr (Fig. b)
after isotope injection. Fewer silver grains are visible over the osteoid
of these rats (b) compared to the pattern seen in the non-diabetic
controls. x 600.
Fig. 8a,b. Autoradiographs of osteoblasts (arrowheads) in CMTtreated diabetic rats a t 20 min (Fig. a) and 4 hr (Fig. b) after isotope
injection. Silver grains can be seen over the osteoblasts (a) and over
the osteoid (b). x 600.
Alkaline Phosphatase (ALPase)
In the control rats, a n intense enzymatic reaction for
ALPase was demonstrated as electron-dense precipitates of lead phosphates along the extracellular aspects
of the plasma membranes of the osteoblasts (Fig. 9).
The cell processes penetrating the osteoid also exhibited a strong enzymatic reaction. The reaction products
Figs. 9-1 2. Ultracytochemical localization of ALPase activity along the plasma membranes of osteoblasts and/or bone-lining cells in the non-diabetic control (9), untreated diabetic (lo), and MC-treated
(111, and CMT-treated diabetic rats (12). Enzymatic reaction can be seen as electron-dense precipitates
of lead phosphates a t the extracellular site of the plasma membranes. Unstained sections. x 10,000.
were also diffusely distributed in the osteoid layer close
to the osteoblast-plasma membranes at the osseous cell
surfaces (Fig. 9). Cytochemical controls demonstrated
that the enzymatic activity was dependent on the presence of the substrate and sensitive to the enzyme inhibitor levamisole in the incubation medium (data not
The ALPase activity was completely absent from the
flattened bone-lining cells in untreated diabetic rats
(Fig. 10). However, when the diabetic rats were treated
with MC, relatively weak ALPase activity was seen to
be localized along the osseous and lateral cell surfaces
of the flattened and/or oval osteoblasts. Tiny precipitates of enzymatic reaction were also noted in the osteoid layer (Fig. 11). In contrast, the osteoblasts in
CMT-treated diabetic rats exhibited intense ALPase
activity comparable to the pattern seen in osteoblasts
of the control rats (compare Figs. 9 and 12).
The enzymatic reaction of Ca-ATPase activity was
demonstrated along the extracellular aspects of the
plasma membranes of the osseous and lateral cell surfaces of osteoblasts in the control rats (Fig. 13).In cytochemical controls, omission of either substrate or the
enzyme activator CaC1, from the incubation medium
eliminated all evidence of this enzymatic reaction (data
not shown).
No evidence of reaction products could be seen in the
atrophic bone-lining cells of the untreated diabetic rats
(Fig. 14). Although the osteoblasts in MC-treated diabetic rats showed a more normal oval outline, they did
not exhibit Ca-ATPase activity (Fig. 15). However, the
osteoblasts in the CMT-treated diabetic rats showed
intense Ca-ATPase reaction a s seen in the control rats
(Fig. 16).
Structural and Cytochemical Changes of Osteoblasts
During Experimental Diabetes
Osteoporosis (osteopenia) has been described a s a
complication of diabetes mellitus in humans and animals (Deleew and Abs, 1975, 1977; Levin et al., 1976;
McNair e t al., 1979; Ramamurthy e t al., 1973; Santiago et al., 1977). Although the mechanisms responsible for this disorder of the skeletal tissues are not yet
known, i t is generally agreed that decreased bone formation rather than accelerated osteoclastic bone resorption is a key factor (Shires et al., 1981). As a result
of inducing diabetes, the unmineralized osteoid layer
separating the mineralized bone from the osteoblast
layer was dramatically reduced in volume, and cuboidal active osteoblasts appeared to be replaced by inactive flattened bone-lining cells, which were mostly devoid of cytoplasmic organelles necessary for protein
synthesis (Golgi apparatus and RER) and active transport (mitochondria). In fact, these bone-lining cells incorporated little 3H-proline and secreted almost no labeled proteins. Mitochondria are involved in part in
regulation of Ca-ATPase activity and cytosolic calcium
concentration (Borle, 1973). Such bone-lining cells are
deficient in cytoplasmic organelles and seem to be functionally inactive, since they produce little procollagen
and lack both Ca-ATPase and ALPase activities, all
being necessary for matrix formation and mineralization.
These osteoblastic functions are regulated by la,25dihydroxyvitamin D, (1,25(OH),D,), and acute and
chronic streptozotocin (STZI-induced diabetes has been
shown to decrease plasma 1,25(OH),D3 levels
(Schneider e t al., 1977; Schedl et al., 1978; Hough et
al., 1981;Shires e t al., 1981; Ishida et al., 1985). Schedl
et al. (1978) suggested that a primary result of insulin
deficiency in diabetes is a n abnormality in vitamin D,
metabolism leading to impaired 1,25(OH),D, production. We recently found that, in the STZ-induced diabetic rat, osteoclasts lost their ruff led border-clear zone
complex (Kaneko et al., 1990). This osteoclast abnormality in diabetes may result from a prior inactivation
of the osteoblasts and decreased release of soluble cytokine(s) by these cells required for osteoclast differentiation (Heath et al., 1985; McSheehy and Chambers,
1986a,b; Thomson e t al., 1986). These results are consistent with earlier reports that diabetes-induced bone
loss (osteopenia) results from impaired bone formation
rather than increased osteoclastic bone resorption
(Hough et al., 1981; Shires et al., 1981; Golub et al.,
1990; Kaneko et al., 1990).
Several possibilities may account for the above findings. Because insulin can increase the osteoblastic production of proteoglycans and collagen, the latter by increasing procollagen mRNA possibly by increasing its
stability and half-life (Craig et al., 1989; Kream et al.,
1985; Weiss et al., 1981), insulin-deficiency diabetes
may inhibit the production of new matrix constituents
by the atrophic osteoblasts (Klein et al., 1985; Schneir
e t al., 1981). This inhibition of matrix formation was
clearly confirmed in this morphometric study. A deficiency of insulin-like growth factor-1 (IGF-1) may also
contribute to bone deficiency due to suppressed bone
formation (Scheiwiller et al., 1986), although it has
recently been shown that the administration of IGF-1
to diabetic rats does not restore bone growth and density to the same extent a s insulin therapy (Binz et al.,
1990). Another possibility is that the osteoid deficiency
might result from pathologically excessive collagenase
activity, which would be expected to accelarate the degradation of newly synthesized bone collagen (Cowen et
al., 1985; Delaisse et al., 1985). Such a n effect would
also be consistent with the elevated collagenase activity seen in rat skin and gingiva during diabetes (Mohanam and Bose, 1983; Ramamurthy and Golub, 1983).
Although collagenase in bone is now known to originate from osteoblasts, not osteoclasts (Sakamoto and
Sakamoto, 1984), and although osteoblasts also produce plasminogen activator that can contribute to the
activation of latent collagenase (Hamilton e t al., 19841,
it is questionable whether the atrophic osteoblasts in
the diabetic animals secrete increased levels of these
proteins (enzymes) to excessively resorb the osteoid. At
the present time, the relative contribution of decreased
protein synthesis and increased extracellular collagen
degradation to the loss of osteoid in the diabetic state
cannot be assessed.
The Effects of Antibiotic and Non-Antibiotic Tetracyclines
on Osteoblasts in Diabetes-Induced Bone Disease
In preliminary studies, we have reported that tetracycline administration can prevent the development of
Figs. 13-1 6. Ultracytochemical localization of Ca-ATPase along the plasma membranes of osteoblasts
and/or bone-lining cells in the non-diabetic control (13),untreated diabetic (14),
and MC-treated (15),and
CMT-treated diabetic rats (16).Unstained sections. X 10,000.
osteopenia in long bones of streptozotocin-induced, insulin-deficient, diabetic rats (Golub et al., 1990). The
oral administration of doxycycline, a semi-synthetic
antimicrobial tetracycline, to these diabetic rats appeared to increase the abnormally deficient bone mass,
bone density, and mineral and organic constituents
even though the severity of hyperglycemia was unaffected by the drug treatment (Golub e t al., 1990). The
current study suggests that minocycline (MC) and its
chemically modified non-antibiotic analogue (CMT)
may ameliorate abnormalities in both osteoblast structure and function in diabetes-induced disease in mature rats. In diabetic rats treated with either MC or
CMT: (1) the osteoid volume was much greater than
that seen in untreated diabetic rats, (2) osteoblasts
showed cuboidal shape with cytoplasmic organization
similar to that in control rats, (3)the osteoblasts incorporated 3H-proline similar to those in control rats, and
(4) the osteoblasts in CMT-treated rats also exhibited
near-normal enzymatic activities of ALPase and CaATPase, which were severely deficient in diabetic osteoblasts (and less severely deficient in MC-treated diabetics). Only Ca-ATPase activity could not be detected
in the osteoblasts in MC-treated diabetics. Although
the MC-treated and CMT-treated diabetic rats were
administered the same oral dose (20 mg per day) of
these drugs, it is possible that the blood levels of the
drugs resulting from these two treatments were different. In this regard, Yu e t al. (1990) recently demonstrated that the oral administration of CMT produced
much higher blood levels than tetracycline-HC1. Thus
it is suggested that CMT is absorbed more efficiently
than MC as well.
From the present study, it cannot be determined
whether these similarities in cytological features of osteoblasts in normal and MC- or CMT-treated diabetic
bones reflect a prevention of the development of diabetic changes or a return of diabetes-induced abnormalities to a more normal state. Another question currently being addressed is whether these antibiotics
(which have long been known to bind metal ions) restore bone cell function by modulating cytoplasmic calcium levels; preliminary studies suggest t h a t this is
possible a t least for osteoclasts (Rifkin et al., 1991).
Consistent with the promotion of osteoid production
in the mature animal by MC or CMT administration,
Polson e t al. (1989) described a significantly increased
deposition of bone matrix in the marrow spaces of the
mandible of adult squirrel monkeys administered tetracycline. Schneir et al. (1990) also reported that MC
administration increased the production of collagen in
the atrophic skin of streptozotocin-diabetic rats. However, the drug’s ability to increase osteoid deposition in
the diabetic’s skeletal tissue could also reflect the inhibition of osteoblast collagenase and a resultant decreased rate of degradation of newly secreted bone collagen. In fact, the in vivo tetracycline administration
to STZ-induced diabetic rats reduced the pathologically-excessive collagenase activity in skin and gingiva
and prevented the loss of skin collagen mass (Golub et
al., 1983, 1984, 1987). Ramamurthy e t al. (1990) further demonstrated that tetracyclines can inhibit the
collagenase secreted by osteoblasts in culture. In summary, it is suggested that (1) diabetes-induced bone
loss (osteopenia) results from decreased protein syn-
thetic and phosphatase activities in osteoblasts and (2)
tetracycline administration is effective in preventing
(or reversing ?) this bone cell abnormality.
This study was supported in part by a grant #DE03987 from the National Institute of Dental Research
(NIH).The authors thank Ms. T. Shiraishi for her technical assistance.
Akisaka, T., T. Yamamoto, and C.V. Gay 1988 Ultracytochemical investigation of calcium-activated adenosine triphosphatase (CaATPase) in chick tibia. J. Bone Mineral Res., 3t19-25.
Ando, T., K. Fujimoto, H. Mayahara, H. Miyayama, and K Ogawa
1981 A new one-step method for the histochemistry and cytochemistry of Ca-ATPase activity. Acta Histochem. Cytochem.,
Binz, K., E.B. Hunziker, C. Schmid, and E.R. Frosch 1990 Osteoporosis in adult streptozotocin diabetic rats is cured by insulin but not
by insulin-like growth factor I (IGF-I). Transact. Orthopaed. Res.
SOC.,36th Ann. Meeting, New Orleans, p. 566.
Boothe, J.H., G.E. Bonvicino, C.W. Waller, J.P. Petisi, R.W. Wilkinson, and R.B. Broschard 1958 Chemistry of the tetracycline antibiotics. I. Quaternary derivatives. J. Am. Chem. SOC.,80:16541657.
Borle, A.B. 1973 Calcium metabolism a t the cellular level. Fed. Proc.,
Cowen, K.S., M. Sakamoto, and S. Sakamoto 1985 Monensin inhibits
collagenase production to osteoblastic cell cultures, and also inhibits both collagenase release and bone resorption in mouse calvaria cultures. Biochem. Int., llr273-280.
Craig, R.G., D.W. Rowe, D.N. Petersen, and B.E. Kream 1989 Insulin
increases the steady state level of a-l(I)procollagen mRNA in the
osteoblast-rich segment of fetal rat calvaria. Endocrinology, 125:
Delaisse, J.M., Y. Eeckhout, C. Sear, A. Galloway, K. McCullah, and
G. Vaes 1985 A new synthetic inhibitor of mammalian tissue
collagenase inhibits bone resorption in culture. Biochem. Biophys. Res. Commun., 133r483-490.
Deleew, I.H., and R. Abs 1975 Bone mass in diabetic. Diabetologia,
Deleew, I.H., and R. Abs 1977 Bone mass and bone density in maturity-type diabetics measured by the lz5I photon-absorption technique. Diabetes, 26:1130-1135.
Fleisch, H., and W.F. Neiman 1961 Mechanism of calcification: role of
collagen, polyphosphate and phosphatase. Am. J . Physiol., 200:
Frank, R.M., and P. Frank 1969 Autoradiographie quantitative de
l’osteogenese en microscopie electronique a l’aide de proline tritiee. Z. Zellforsch 99,121-133.
Frauran-Clavel, M.J., and J Oustrin 1986 Alkaline phosphatase and
bone calcium parameters. Bone, 7t95-99.
Fukushima, O., and N. Goshi 1983 Neutral ATP-hydrolyzing enzyme
activity on the plasma membrane of osteoblasts. Acta Histochem.
Cytochem., 16r216-222.
Golub, L.M., R.A. Greenwald, E.J. Zebrowski, and N.S. Ramamurthy
1978 The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen. Biochim. Biophys.
Acta, 534:78-81.
Golub, L.M., T.F. McNamara, G. DAngelo, R.A. Greenwald, and N.S.
Ramamurthy 1987 A non-antibacterial chemically-modified tetracycline inhibits mammalian collagenase activity. J. Dent. Res.,
Golub, L.M., N.S. Ramamurthy, H. Kaneko, T. Sasaki, B. Rifkin, and
T.F. McNamara 1990 Tetracycline administration prevents diabetes-induced osteopenia in the rats: Initial observations. Res.
Commun. Chem. Pathol. Pharmacol., 68:27-40.
Golub, L.M., H.M. Lee, G. Lehrer, A. Nemiroff, T.F. McNamara, R.
Kaplan, and N.S. Ramamurthy 1983 Minocycline reduces gingival collagendlytic activity during diabetes: preliminary observations and a proposed new mechanism of action. J. Periodont. Res.,
Golub, L.M., N. Ramamurthy, T.F. McNamara, B. Gomes, M. Wolff,
A. Casino, A. Kapoor, J. Zambon, S. Ciancio, M. Schneir, and H.
Perry 1984 Tetracyclines inhibit tissue collagenase activity. A
new mechanism in the treatment of periodontal disease. J. Periodont. Res., 19:651-655.
Games, B.C., L.M. Goluh, and N.S. Ramamurthy 1984 Tetracyclines
inhibit parathyroid hormone-induced bone resorption in organ
culture. Experientia, 40t1273-1275.
Hamilton, J.A., S.R. Lingelhach, N.C. Partridge, and T.J. Martin 1984
Stimulation of plasminogen activator in osteohlast-like cells by
hone-resorbing hormones. Biochem. Biophys. Res. Commun., 122;
230-236, 1984.
Heath, J.K., J. Saklatvala, M.C. Meikle, S.J. Atkinson, and J.J. Reynolds 1985 Pig interleukin l(cataho1in)is a potent stimulator of
bone resorption in vitro. Calcif. Tissue Int., 37:95-97.
Hough, S., L.V. Avioli, M.A. Bergfeld, M.D. Fallon, E. Slatopolsky,
and S.L. Teitelhaum 1981 Correction of abnormal bone and mineral metabolism in chronic streptozotocin-induced diabetes mellitus in the rat by insulin therapy. Endocrinology, 108t22282234.
Ishida, H., Y. Seino, S. Nishi, N.Kitano, M. Seno, T. Taminato, and S.
Matsukura 1985 Effects of insulin on altered mineral and vitamin D metabolism in streptozotocin-induced diabetes. Acta Endocrinologica, 108t231-236.
Jaffe, N.R. 1976 Alkaline phosphatase activity, characterization, and
suhcellular distribution during initial skeletogenesis in the prenatal rat limb. Calcif. Tissue Res., 21:153-162.
Kaneko, H., T. Sasaki, N. Ramamurthy, and L.M. Golub 1990 Tetracycline administration normalizes the structure and acid phosphatase activity of osteoclasts in streptozotocin-induced diabetic
rats. Anat. Rec., 227:427-436.
Klein. L.. X.I. Ying-Yan, and K.M. Wong 1985 Effect of short-term
streptozotocin-Ynduced diabetes on b&e resorption, mineralization and collagen formation in the rat. Calcif. Tissue Int., 37:ahst.
Kream, B.E., M.D. Smith, E. Canalis, and L.G. Raisz 1985 Characterization of the effect of insulin on collagen synthesis in fetal rat
bone. Endocrinology, 116t296-302.
Levin, M.E., V.S. Boisseau, and L.V. Avioli 1976 Effects of diabetes
militus on hone mass in juvenile and adult-onset diabetes. N.
Engl. J . Med., 294t241-245.
McCormick, J.R.D., S.M. Fox, L.L. Smith, B.A. Bitler, J. Reichenthal,
V.E. Origoni, W.H. Muller, R. Winterbottom, and A.P. Doerschuk
1957 Studies of the reversible epimerization occurring in the tetracycline family. The preparation, properties and proof of structure of some 4-epi-tetracyclines. J. Am. Chem. SOC.,79t28482859.
McNair, P., S. Madshad, C. Christiansen, M.S. Christiansen, O.K.
Faher, C. Binder, and I. Transbo 1979 Bone loss in diabetes: effects of metabolic state. Diahetologia, 17t283-286.
McSheehy, P.M.J., and T.J. Chambers 1986a Osteohlastic cells mediate osteoclastic responsiveness to parathyroid hormone. Endocrinology, 118t824-828.
McSheehy, P.M.J., and T.J. Chambers 198613 Osteoblast-like cells in
the presence of parathyroid hormone release soluble factor that
stimulates osteoclastic bone resorption. Endocrinology, 119:
Mayahara, H., H. Hirano, T. Saito, and K. Ogawa 1967 The new lead
citrate method for the ultracytochemical demonstration of nonspecific alkaline phosphatase. Histochemie, 1lt88-96.
Melancon, M.J., and H.F. DeLuca 1970 Vitamin D stimulation of
calcium-dependent adenosine triphosphatase in chick intestinal
brush borders. Biochemistry 9,1658-1664.
Mohanan, S., and S.M. Bose 1983 Influence of streptozotocin and alloxan induced diabetes in the rat on collagenase and certain lysosomal enzymes in relation to the degradation of connective tissue proteins. Diahetologia, 25t66-70.
Niiweide. P.J.. E.W.M. Kawilarane de Haas. and A.M. Wassenaar
198f Alkaline phosphatase a& calcification, correlated or not?
Metah. Bone Dis. Rel. Res., 3:61-66.
Polson, A.M., O.J. Bouwsma, T.F. McNamara, and L.M. Golub 1990
Enhancement of normal hone density after tetracycline administration. J. Periodontal., (in press).
Ramamurthy, N.S., and L.M. Goluh 1983 Diabetes increases collagenase activity in extracts of rat gingiva and skin. J . Periodont.
Res., 17t455-462.
Ramamurthy, N.S., E.J. Zehrowski, and L.M. Golub 1973 Alloxan
diabetes and reduced hone density in rat mandible. Res. Commun.
Chem. Pathol. Pharmacol., 5514-619.
Ramamurthy, N.S., A.T. Vernillo, H. Lee, L.M. Goluh, and B. Rifkin
1990 Tetracyclines inhibit collagenase from osteohlastic rat UMR
cells. J. Dent. Res., 69(Sp 1ss):Ahstr. 1932, p. 350.
Register, T.C., F.M. McLean, M.G. Low, and R.W. Wuthier 1986 Roles
of alkaline phosphatase and labile internal mineral in matrix
vesicle-mediated calcification. J. Biol. Chem., 261t9354-9360.
Rifkin, B., H. Donahue, K. Iijima, M. Oligorsky, and C. Ruhin 1991
Tetracyclines attenuate extracellular calcium-stimulated cytosolic calcium mobilization in osteoclasts. J. Dent. Res., 70(Sp Iss):
ahst. (in press).
Rifiin, B., F. Sanavi, A. Kleckner, B. Games, N.S. Ramamurthy, and
L.M. Goluh 1988 Nan-antibacterial tetracycline inhibits hone resorption in organ culture. J. Dent. Res., 67(Sp Iss): ahst. 2068.
Sakamoto, S., and M. Sakamoto 1984 Osteohlast collagenase: Collagenase synthesis by clonally derived mouse osteogenic (MC3T3E l ) cells. Biochem. Internat., 951-58.
Santiago, J.V., W.H. McAlister, S.K. Ratzan, Y. Bussman, M.W. Haymond, G. Shakelford, and V.V. Weldon 1977 Decreased cortical
thickness and osteopenia in children with diabetes mellitus. Clin.
Endocrinol. Metah., 45:845-848.
Schedl, H.P., H. Heath, and J . Wenger 1978 Serum calcitonin and
parathyroid hormone in experimental diabetes: Effects of insulin
treatment. Endocrinology, 103t1368-1373.
Scheiwiller, E., H-P. Guler, J. Merryweather, C. Scandella, W. Maerki, J. Zapf, and E.R. Froesch 1986 Growth restoration of insulindeficient diabetic rats by recombinant human insulin-like growth
factor I. Nature 323t169-171.
Schneider, L.E., H.P. Schedl, T. McCain, and M.R. Haussler 1977
Experimental diabetes reduces circulating 1,25-dihydroxyvitamin D in the rat. Science, 196:1452-1454.
Schneir, M., N. Ramamurthy, and L.M. Goluh 1981 Diabetes reducesmandibular collagen synthesis rather than enhancing its cataholism. J. Dent. Res., 60 (Sp 1ss):AGO: 1332.
Schneir, M., N.S. Ramamurthy, and L.M. Goluh 1990 Minocycline
treatment of diabetic rats normalizes skin collagen production
and mass. Matrix: Collagen Rel. Res., 10t112-123.
Shen, V., K. Hruska, and L.V. Avioli 1988 Characterization of a (Ca,
Mg)-ATPase system in the osteohlast plasma membrane. Bone,
Shires, R., S.L. Teitelbaum, M.A. Bergfeld, M.D. Fallon, E. Slatopolsky, and L.V. Avioli 1981 The effect of streptozotocin-induced
chronic diabetes mellitus on hone and mineral homeostasis in the
rat. J. Lab. Clin. Med., 97:231-240.
Takagi, M., R.T. Parmley, Y. Toda, and F.R. Denys 1983 Ultrastructural cytochemistry of complex carbohydrates in osteohlasts, osteoid, and hone matrix. Calcif. Tissue Int., 35t309-319.
Thomson, B.M., J. Saklatvala, and T.J. Chambers 1986 Osteohlasts
mediate interleukin 1 stimulation of hone resorption by rat osteoclasts. J. Exp. Med., 164t104-112.
Weiss, R.E., A.H. Gorn, and M.E. Nimni 1981 Abnormalities in the
biosynthesis of cartilage and hone proteoglycans in experimental
diabetes. Diabetes, 3Ot670-677.
Wlodarski, K.H., and A.H. Reddi 1986 Alkaline phosphatase a s a
marker of osteoinductive cells. Calcif. Tissue Int., 39t283-285.
Wright, G.M., and C.P. Lehlond 1981 Immunohistochemical localization of procollagens. J . Histochem. Cytochem., 29:791-804.
Yoshiki, S., T. Umeda, and Y. Kurahashi 1972 An effective reactivation of alkaline phosphatase in hard tissues completely decalcified for light and electron microscopy. Histochemie, 29t296-304.
Yu, Z., M. Leung, N.S. Ramamurthy, T. McNamara, and L.M. Golub
1990 Serum levels of chemically-modified tetracycline: A comparison to tetracycline. J. Dent. Res., 69(Sp Iss): abstr. 1092, p. 245.
Без категории
Размер файла
1 418 Кб
experimentov, structure, diabetes, administration, tetracyclic, osteoblastic, restore, function
Пожаловаться на содержимое документа